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THE PROPAGATION, CHARACTERISATION AND 
OPTIMISATION OF CANNABIS SATIVA L 
AS A PHYTOPHARMACEUTICAL 



A thesis submitted by 
David Potter J P 
MIBiol CBiol FLS CMIOSH 



In fulfilment of the requirement 
for the degree of Doctor of Philosophy (PhD) 
in Pharmaceutical Sciences 



Department of Pharmaceutical Science Research 
King's College London 



March 2009 



Abstract 

In response to known pharmacology, and an increasing weight of anecdotal evidence of 
efficacy, clinical trials have been performed to support the licensing of cannabis-based 
botanical medicines. The initial applications envisaged were the treatment of cancer 
pain, neuropathic pain and various symptoms associated with multiple sclerosis. With 
effective alternatives often unavailable, otherwise law-abiding UK patients have regularly 
turned to illicit cannabis for medical relief. The main active ingredients in this are the 
cannabinoids THC and CBD, but other pharmacologically active cannabinoids are also 
present. One study reported here quantifies these cannabinoids and assesses the likely 
implications for efficacy. Using light microscopy, studies are performed to expand current 
knowledge of the form and function of trichomes in Cannabis sativa L. Supporting 
chemical analyses ascertain what secondary metabolites are biosynthesised within 
these trichomes, and determines where and when this occurs. To comply with the 
demands of the pharmaceutical industry, and in marked contrast to illicit cannabis, a 
phytopharmaceutical feedstock must meet high expectations regarding the minimum 
and maximum content of a range of compounds. Specific studies are performed to 
ascertain how growing methods affect the secondary metabolite content. They also aim 
to find out how a tight specification can be met while satisfying commercial and 
environmental expectations. This involves studying plant development and secondary 
metabolite biosynthesis in both indoor and outdoor conditions. The first approved 
cannabis-based botanical medicine supported by this research is Sativex®. This 
became available in Canada in 2005 for the treatment of central neuropathic pain in 
multiple sclerosis and in 2007 for intractable cancer pain. The medicine is also available 
in the UK and many other countries on a 'named patient basis'. This thesis has also 
supported the production of a range of other cannabinoids which are undergoing in-vitro 
and in-vivo testing. This could lead to the commercial production of an increasing range 
of phytopharmaceuticals. 



Cannabis sativa L cv Gayle 
CBD Chemotype. 
Awarded European Plant Variety Rights EU 16301, 10 th Oct 2005 



<7o my best mate John (Poof^ 
and those who cared for him at 
St Leonards J-Cospice, Yorf^ 



ii 



Acknowledgements 

This PhD would not have been possible without the support of friends and colleagues, of 
which there are too many to comprehensively mention. My deep appreciation first goes 
to my dear friend and champion Dr Brian Whittle who, having convinced me to do the 
PhD, guided me on my 'long road to Ithaka'. Enormous thanks goes too to my supervisor 
Professor Marc Brown for his faith in me and his support and patience throughout. 
I express my sincerest gratitude to GW Pharmaceuticals Ltd, particularly Dr Geoffrey 
Guy, for sponsoring this PhD. Special thanks goes to a fabulous team of botanical 
colleagues - Dr Etienne de Meijer, DrTim Wilkinson, Barry and Chris Mears, Keith Hines 
and Roger Phillips for supplying cannabis and moral support throughout the project. I am 
especially grateful to Kathy Hammond for diligently analysing so many of my samples 
and Heather North for administrative assistance and pastoral care. 

Down on the farm I am indebted to great friends John and Vicky Clinch, over whose 
kitchen table much of the PhD was planned. In addition to growing many of my crops, 
they assisted in overnight monitoring as plants were dried. In the interest of science 
they also tolerated the incredible mess as I made CBD-rich hashish in their kitchen. 

Aren't our police wonderful? Much of the work would not have been possible without help 
from several constabularies, and I would like to thank officers Phil Painter, Jerry Prodger, 
Howard Chandler, Bill Stupples and Steve Holme for assisting me with my enquiries. 
Heartfelt thanks goes to an ex-colleague and fond friend Heather House, who was my 
co-speaker at several inspirational Multiple Sclerosis Group Meetings. My thanks go to 
Dr Peter Toomey for his services to St Leonards Hospice in York, and for the invitation to 
give a presentation at a memorable Yorkshire Pain Management Group Meeting at 
St Stephens College - an experience that opened my eyes to the dedicated work of 
those involved in pain control. Warm thanks go too to good friends Peter Smith for 
photographic assistance and Valerie Bolas for botanical illustrations, and to colleagues 
at Kings College London - especially Darragh Murnane and Yanjun Xhao. Finally, I thank 
my wife Jane for supporting my PhD application - and for her forebearance thereafter. 

iii 



List of Publications 

Potter, D.J., 2004. Growth and Morphology of Medicinal Cannabis. In Guy, G.W., 
Whittle, B.A. and Robson, P.J. (Eds.). The Medicinal Uses of Cannabis and 
Cannabinoids. Pharmaceutical Press, London, pp. 17-54. 

Potter, D.J., Clark, P. and Brown M.B. 2008. Potency of A 9 -THC and other cannabinoids 
in cannabis in England in 2005: Implications for psychoactivity and pharmacology. 
J Forensic Sci 53 (1): 90-94. 

Russo, E.B., Jiang, H-E., Li, X., Sutton, A., Carboni, A., del Bianco, F., Mandolino, G., 
Potter, D.J., Zhao, Y.X., Bera, S., Zhang, Y-B., Lu, E-G., Ferguson, D.K., Hueber, F., 
Zhei, L-C, Liu, C-J., Wang Y-F., Li C-S. 2008. Phytochemical and genetic analyses of 
ancient cannabis from Central Asia. J. Exp. Bot. 59, 15, 4171-4182. 



iv 



Table of Contents 

Abstract i 

Acknowlegements iii 

List of Publications iv 

Table of Contents v 

List of Figures xiii 

List of Tables xxii 

Abbreviations xxvii 

Chapter 1 INTRODUCTION 1 

1.1 Plants as a source of medicines - past and presen 1 

1.2 Cannabis Botany 4 

1 .3 Cannabis Taxonomy 6 

1 .4 UK Medicinal Cannabis Use - History and Legal Complications 8 

1 .5 The influence of the BMA and the House on Lords Select Committee on 
Science and Technology on UK Cannabis Research 10 

1.6 International Legal Attitudes to Medicinal Cannabis 11 

1.6.1USA 12 

1.6.2 Canada 14 

1.6.3 Mainland Europe 14 

1.6.4 Ireland 15 

1.6.5 Australia 15 

1.6.6 Japan 15 

1.7 The choice of active pharmaceutical ingredients (APIs) 15 

1.8 Cannabinoid and terpene biosynthesis 17 

1.9 Cannabinoid Receptors and Cannabinoid Pharmacology 21 

1.10 Outline of Thesis 24 



V 



CHAPTER 2 CHARACTERISATION OF ILLICIT CANNABIS IN THE UK 27 

2.1 INTRODUCTION 27 

2.2 AIM AND OBJECTIVES 29 

2.3 MATERIALS 29 

2.3.1 Cannabis samples 29 

2.3.2 Microscopy, Photography and other Apparatus 29 

2.4 METHODS 30 

2.4.1 Collection of Representative Samples 30 

2.4.2 Storage of illicit cannabis samples 30 

2.4.3 Categorisation of the form of each sample 30 

2.4.3 Categorisation of the form of each sample 30 

2.4.3.1 Cannabis resin 30 

2.4.3.2 Herbal cannabis 31 

2.4.3.3 Sinsemilla 31 

2.4.3.4 Cannabis powder 32 

2.4.3.5 Other categories not included 33 

2.4.4 Measurement of cannabinoid potency and profile 33 

2.4.5 Statistical Analysis 33 

2.5 RESULTS AND DISCUSSION 34 

2.5.1 Categorisation of cannabis type between regions 34 

2.5.2 The range of cannabinoids in each cannabis category 34 

2.5.3 Comparison of cannabis potency and profile between regions 38 

2.5.4 Trends in Cannabis Potency 39 

2.5.5 The efficacy of illicit cannabis 42 

2.6 CONCLUSIONS 45 

Chapter 3 Cannabis trichome form, function, and distribution 47 

3.1 INTRODUCTION 47 

3.2 AIM AND OBJECTIVES 49 

vi 



3.3 MATERIALS 50 

3.3.1 Germplasm 50 

3.3.2 Microscopy, Tissue Stains, Photography and other Apparatus 51 

3.4 METHODS 51 

3.4.1 Photomicrograph Studies 51 

3.4.1.1 Choice of Microscopes 51 

3.4.1.2 Staining 52 

3.4.1.3 Unmounted Sample Preparation 52 

3.4.1.4 Mounted sample preparation 52 

3.4.1.5 Illumination 53 

3.4.1.6 Photography 53 

3.4.1.7 Isolation and Observation of Detached Glandular Resin Heads 54 

3.4.2 Effect of glandular trichome array on the secondary metabolite content 

of plant tissues 54 

3.4.3 Organoleptic Assessment of the Effect of Trichome Colour and 
Pubescence Density on cannabis potency 55 

3.4.4 Effect of photosynthetic ability, or lack of ability, on cannabinoid 
biosynthesis in sessile trichomes 56 

3.4.5 Statistical Methods 57 

3.5 RESULTS AND DISCUSSION 57 

3.5.1 Photomicrograph studies 57 

3.5.1 .1 Simple unicellular trichomes 57 

3.5.1 .2 Cystolythic trichomes 58 

3.5.1 .3 Capitate sessile trichomes 59 

3.5.1 .4 Antherial Sessile Trichomes 60 

3.5.1 .5 Capitate Stalked Trichomes 61 

3.5.1 .6 Bulbous Trichomes 73 

3.5.1 .7 Effect of age and storage on glandular trichome colour 73 

3.5.2 Effect of glandular trichome array on the secondary metabolite content 

of plant tissues 74 

vii 



3.5.3 Effect of capitate stalked trichome density and colour on cannabinoid 
content and profile 76 

3.5.4 Effect of photosynthetic ability, or lack of ability, on cannabinoid 
biosynthesis in sessile trichomes on variegated leaf tissue 80 

3.6 CONCLUSIONS 82 

Chapter 4. The Function and Exploitation of Secondary Metabolites from 

Glandular Trichomes of Cannabis sativa L 84 

4.1 INTRODUCTION 84 

4.2 AIM AND OBJECTIVES 86 

4.3 MATERIALS 87 

4.3.1 Germplasm 87 

4.3.2 Apparatus 87 

4.4 METHODS 87 

4.4.1 Separation of Sessile and Capitate Stalked Trichomes Glandular 
Heads from mature fresh floral material 87 

4.4.2 Bulk-Production of Pure Sessile Trichome Preparations 88 

4.4.3 Production of a cannabichromene-rich sessile trichome preparation 88 

4.4.4 Ontogenetic changes in Secondary Metabolite Content of Glandular 
Trichome Contents 88 

4.5 RESULTS AND DISCUSSION 90 

4.5.1 Separation of Sessile and Capitate Stalked Trichomes Glandular 
Heads from mature fresh floral material 90 

4.5.2 Isolation of intact sessile glandular trichomes from vegetative material. 91 

4.5.3 The collection of sessile trichomes from foliage of a high CBC 
chemotype as a means of isolating the minor cannabinoid CBC 94 

4.5.4 Ontogenetic changes in glandular trichome secondary metabolite 
content 94 

4.6. CONCLUSIONS 100 



viii 



Chapter 5 Indoor Propagation of Medicinal Cannabis 102 

5.1 INTRODUCTION 102 

5.2 AIM AND OBJECTIVES 106 

5.3 MATERIALS 107 

5.3.1 Plant Propagation and Drying Materials 107 

5.3.2 Germplasm Details 108 

5.3.3 Light Measurement and Weighing Equipment 108 

5.3.4 Growth Medium 108 

5.4 METHODS 109 

5.4.1 Routine Propagation and Plant Production Methods 109 

5.4.1.1 Seed sowing and transplantation of seedlings 109 

5.4.1.2 Production of Cuttings (Clones) 109 

5.4.1.3 Nurturing Vegetative Growth of Seedlings and Cuttings 110 

5.4.1.4 Induction and Maintenance of Flowering 110 

5.4.1.5 Biological Pest Control 110 

5.4.1.6 Harvesting 110 

5.4.1.7 Crop Drying 111 

5.4.1.8 Stripping 112 

5.4.1.9 Garbling 112 

5.4.1.10 Storage 112 

5.4.1.11 Environmental Control System 113 

5.4.2 Specific Methods 113 

5.4.2.1 Uniformity of Plants Grown from Cuttings or Seed 113 

5.4.2.2 Effect of Duration of Flowering Period on Yield 1 14 

5.4.2.3 Effect of Daylength on Cannabinoid Profile (Part 1) Comparison of 
12 and 13 hour daylength 114 

5.4.2.4 Effect of Daylength on Cannabinoid Profile (Part 2) Comparison of 

1 1 and 1 2 hour daylength 1 1 5 

5.4.2.5 Plant height assessment 115 

5.4.2.6 Stigma senescence assessment 116 

ix 



5.4.2.7 Plant Weight Assessment 116 

5.4.2.8 Cannabinoid Content and Profile 116 

5.4.2.9 Effect of Irradiance Level on Plant and Cannabinoid Yield 117 

5.4.2.10 Effect of the length of flowering period on the cannabinoid profile 

of heterozygous plants of the mixed THC/CBD chemotype 118 

5.4.2.11 Statistical Analysis 118 

5.5 RESULTS AND DISCUSSION 119 

5.5.1 Comparison of the Yield and Uniformity of Plants Grown from 
Cuttings or Seeds 119 

5.5.2 Effect of Irradiance Level on Plant and Cannabinoid Yield 120 

5.5.3 Effect of Duration of Flowering Period on BRM and Cannabinoid 
Yield 126 

5.5.3.1 Effect of Duration of Harvest Period on Ratio of THC and CBG in 
THC Chemovars 127 

5.5.3.2 Effect of the length of flowering period on profile of heterozygous 
chemotypes with mixed THC/CBD profiles 129 

5.5.4 Effect of Daylength on Plant Development and Cannabinoid Profile ...130 

5.5.4.1 Comparison of Twelve and Thirteen Hour Daylength Regimes 130 

5.5.4.2 Comparison of Eleven and Twelve Hour Daylength Regimes 136 

5.5.4.3 Review of the comparisons of plants induced to flower on 
daylengths 11, 12 and 13 hours 140 

5.6 CONCLUSIONS 141 



Chapter 6 The Outdoor Propagation of Phytopharmaceutical Cannabis 143 

6.1 INTRODUCTION 143 

6.2 AIM and OBJECTIVES 144 

6.2.1 The effect of growing environment on female plant development 145 

6.2.2 Comparison of the secondary metabolite yield and profile of fresh 
plant material and enriched trichome preparations made from them ...145 



6.2.3 The effects of harvest timing on secondary metabolite yield and profile 
145 

6.2.4 Comparison of the secondary metabolite profiles of glasshouse and 
outdoor grown plants 145 

6.2.5 Evaluation of outdoor pest and disease issues 145 

6.2.6 Evaluation of Crop Drying Methods 145 

6.3 MATERIALS 146 

6.4 GENERAL AGRONOMIC METHODS 146 

6.4.1 Seedbed Location, Preparation and Crop Establishment 146 

6.4.2 Field Trial Design 147 

6.4.3 Soil Nutrition 147 

6.4.4 Pest and Disease Monitoring and Management 147 

6.4.5 Harvest 147 

6.4.6 Crop Drying and Stripping 147 

6.4.7 Assessment of Crop Development 148 

6.5 Secondary Metabolite Purification and Analytical Methods 148 

6.5.1 Production and Collection of Enriched Trichome Preparations 148 

6.5.2 Steam distillation of trichome rich preparations 148 

6.5.3 Steam distillation fresh foliar and floral material 149 

6.6 Statistical methods 149 

6.7 RESULTS and DISCUSSION 149 

6.7.1 Observations on Crop Establishment and Plant Development 149 

6.7.2 Comparison of the secondary metabolite yield and profile of fresh 
plant materials and enriched trichome preparations made from them .154 

6.7.3 The effects of harvest timing and growth stage on yield and 
cannabinoid profile 156 

6.7.3.1 Botanical Raw Material Yield 156 

6.7.3.2 Potency of CBD chemovars 157 

6.7.3.3 CBD Yield 158 

xi 



6.7.3.4. Effect of Harvest Date and Growth Stage at Harvest on 

Cannabinoid Profile 159 

6.7.4 Effect of Growth Stage and Harvest Date on Essential Oil Profile 161 

6.7.5 Comparison of the secondary metabolite content of glasshouse and 

outdoor grown plants 165 

6.7.6 Summary of Pest and Disease Problems in the Field Trials 167 

6.7.7 Effect of Raised Temperatures on Crop Drying Time 171 

6.8 CONCLUSIONS 172 

Chapter 7 GENERAL DISCUSSION 174 

REFERENCES 185 

APPENDICES 215 



xii 



List of Figures 



Figure 1.1. Contrasting leaf morphology in three clones of Cannabis sativa L, (a) CBD 
chemotype G5 M16 cv Gill, (b) THC chemotype G1 M3 cv Guinevere (c) Afghan 
landrace clone M146 (Illustrations by Valerie Bolas, commissioned by GW 
Pharmaceuticals. 4 

Figure 1.2. (a) Male (left) and female cannabis (right) in later stage of flowering, (b) 
Female cannabis inflorescence, (c) A cluster of male flowers with sepals split open and 
reflexed to expose the anthers. 5 

Figure 1.3. Biosynthetic pathway of THC and THCV, via CBG or CBGV. 1, 
Geranylpyrophosphate; 2, Divarinic Acid (R1) or Olivetolic Acid (R2); 3, 
Cannabigerovarin (CBGV) (R1) or Cannabigerol (CBG) (R2);. 4, A9- 
tetrahydrocannabivarin (R1) or A9-tetrahydrocannabinol (R2). R1 (-C3H7) and 
R2(-C5H11) indicate the propyl or pentyl forms of the metabolites; Enzyme I: 
geranylpyrophosphate:olivetolate geranyltransferase(GOT); Enzyme II: THC(V) 
synthase. 18 

Figure 1.4. The two pathways of isopentyl diphosphate (IPP) biosynthesis in plants, as 
found in the plastid and cytosol respectively. 20 

Figure 1.5. The disassembly of an activated G-protein into two signalling components. 
(Alberts et al., 2002) 22 

Figure 2.1. Examples of cannabis resin samples (<1g up to 230g) seized by police in 
2004/5. 30 

Figure 2.2. (a) Loose herbal cannabis material showing separated seeds; (b) 
Compressed herbal cannabis material with selection of removed seeds. 31 

Figure 2.3. A typical confiscated sample of illicit sinsemilla cannabis consisting of three 
separate packets, each containing approximately one gram. 32 



Xlll 



Figure 2.4. (a) A herb grinder in closed position; (b) An open herb grinder revealing the 
component parts. 32 

Figure 2.5. (a) The balance of THC, CBD and CBN in sinsemilla (n = 256); (b) The 
balance of THC, CBD and CBN in herbal cannabis (n = 35); (c) The balance of THC, 
CBD and CBN resin (n = 169). 37 

Figure 2.6 The correlation between THC and CBN content in resin samples seized in five 
constabularies in 2004/5 (n = 169). 38 

Figure 2.7. A comparison of the range and distribution patterns of THC content of seized 
imported herbal cannabis samples in 1 998, King et al. (2004) (n = 44) and 2005 Potter et 
al.(2008)(n = 33). 39 

Figure 2.8. A comparison of the ranges of THC contents of Sinsemilla seized in the UK 
and analysed by the Forensic Science Service in 1996-8 (n = 145) and samples seized 
by police in Derbyshire (n=15), Kent (n=58), London Metropolitan (n = 96), Merseyside (n 
= 44) and Sussex (n = 34) in 2004/5 (total n = 247). 40 

Figure 3.1. Upper surface of a bract within a cannabis inflorescence showing glandular 
stalked trichomes to be present only within the proximal region. 55 

Figure 3.9. A variegated leaf of clone M60, with 1cm diameter disks cut from 
symmetrically opposite sides of the midrib. 57 

Figure 3.3. (a) Unicellular non-glandular trichome. The sample is temporarily mounted 
under hemp oil and viewed in transmitted light; (b) Cystolythic trichomes observed on the 
leaf margin of a young leaf. The sample was temporarily dry-mounted and viewed in 
transmitted light. Cystolyths (concretions of calcium carbonate) are visible at the base of 
each trichome. 58 

Figure 3.4. (a) A capitate sessile trichome observed on the edge of one of the first pair of 
true leaves of a cannabis seedling. The specimen was temporarily dry-mounted and 
viewed using both transmitted and incident light; (b) a sessile trichome on a leaf surface 



stained with Fast Blue. The still-wet sample was temporarily dry-mounted and viewed 
using incident light. 59 

Figure 3.5. (a) a row of antherial sessile trichomes showing their normal distribution in 
the furrow of a cannabis anther. These anthers were captured in incident light through a 
low-power microscope, (b) closer view of antherial trichomes. 61 

Figure 3.6. A capitate stalked trichome (centre) between two cystolythic trichomes. The 
specimen is temporarily dry-mounted and illuminated from below. The secretory cells 
are out-of-focus due to the optical distortion within the glandular head. 62 

Figure 3.7. Two dry-mounted capitate stalked trichomes viewed in transmitted light. 
Most of the features are out-of-focus. In the right-hand trichome, a crisp view of cells 
within the secretory cell disk appears as an in-focus image. However these appear to 
be located outside of the trichome structure, due to the refractive properties of the resin 
head. 63 

Figure 3.8. (a) A temporarily dry-mounted capitate stalked trichome viewed in 
transmitted light. An irregular arrangement of poorly-defined secretory cells is visible at 
the base of the glandular head; (b) A capitate stalked trichome, temporarily mounted in 
glycerol and viewed in transmitted light, and (c) an illustration of a capitate stalked 
trichome on Cannabis sativa by Briosi and Tognini (1894). 64 

Figure 3.9. (a and b). Similar sized capitate stalked trichomes temporarily mounted 
under hemp oil. The samples are viewed in transmitted light. Possibly because of a 
similarity in the refractive index of the oil and the secretory cell contents, these cells 
appear clear. The outer membrane at the base of the glandular head appears dark and 
opaque. 65 

Figure 3.10. A contrasting pair of resin heads on capitate stalked glandular trichomes, 
naturally orientated to allow sideways-on (left) and overhead views (right). The 
specimen is temporarily mounted in a 70% v/v aqueous solution of glycerol and 
illuminated from below. 66 



Figure 3.11. (a) Secretory cells stained red, within the glandular head, after thirty minutes 
in 1% tetrazolium red; (b) A capitate stalked trichome with glandular head removed by 
slight abrasion. After thirty minutes in 1% tetrazolium the disk of secretory cells is 
stained bright red; (c) A mature capitate stalked trichome between two non-glandular 
cystolythic trichomes, viewed after twelve hours in 0.1% tetrazolium solution. 67 

Figure 3.12. (a) A capitate stalked trichome temporarily dry-mounted and viewed in 
transmitted and incident light. The glandular head has become partly detached from the 
stalk to expose the stipe cells, which connect the disk of secretory cells to the 
hypodermal cells within the stalk; (b) The stalk of a capitate stalked trichome after 
detachment of the resin head. The specimen was dry-mounted and illuminated with 
incident light. The stipe cells can just be seen protruding from the top of the stalk. 68 

Figure 3.13. Separation of the glandular head during (left) and after (right) the 
appearance of a fissure above the secretory cells. The example shown was observed 
on (M280). The specimen was viewed in incident light. 68 

Figure 3.14. (a) An intact glandular stalked trichome of a naturally pigmented clone 
M186, with coloured cells visible within the resin head. The sample was temporarily 
mounted in hemp oil; (b) A direct overhead-view of the stalk of a capitate stalked 
trichome on clone M186 after resin head detachment. The resin head has become 
detached leaving thr base of the secretory head attached to the stalk. The sample was 
temporarily mounted in oil. 70 

Figure 3.15. A detached resin head (approximately 100pm diameter) from a capitate 
stalked trichome, viewed from below to gain a clear view of the scar where the stipe cells 
were originally attached. The head had been trapped on the surface of clear adhesive 
tape. 71 

Figure 3.16. (a) A dense pubescence of glandular stalked trichomes on a bract within a 
cannabis female inflorescence. The specimen was illuminated from behind and 
photographed with a tripod-mounted camera incorporating a macro lens. The 
orange/brown structures are senesced stigmas; (b) two young cotton-melon aphids 



XVI 



Aphis gossypii. All six legs on each specimen are irreversibly adhered to the resin heads 
of capitate stalked trichomes. 72 

Figure 3.17. (a) A small bulbous trichome (left) alongside a fully developed glandular 
stalked trichome. The contrast in resin head diameter (10 urn v 100 urn) is clear; (b) a 
simple bulbous trichome and (c) a complex bulbous trichome. These are 10-15 urn in 
diameter. These samples were temporarily dry-mounted and viewed in mixed 
transmitted and incident light. 73 

Figure 3.18. (a) Clear glandular stalked trichomes on freshly harvested young cannabis 
floral tissue; (b) Brown trichomes on three-year old stored cannabis. 74 

Figure 3.19. The mean density of capitate stalked and sessile trichomes (± 1SE) (upper 
and lower surfaces combined), on each of three high-THC cultivars M3, M6 and M7. On 
each clone twenty randomly selected fields were counted on both the upper and lower 
surface in the proximal and distal areas. 74 

Figure 3.20. The proportion of CBC expressed as % of THC+CBC in the cannabinoid 
profile of proximal and distal tissue of bracts from each of three high-THC clones. (Error 
bars on clones represent ± 1SD, and on the mean ± SE). In all three the difference was 
highly significant (ANOVA, *** denotes p < 0.001 ). 75 

Figure 3.21. The correlation between capitate stalked trichome density (visually 
assessed 1-9 scale) and the overall THC content of the sample. Values shown are the 
mean % w/w THC content (± SE only where n > 5) of all samples for each density score. 
Regression line is shown in red. The regression model is:- % THC = 18.051 - 1.629 * 
Density Score. (p< 0.001, R 2 = 0.17). 76 

Figure 3.22. The correlation between capitate stalked trichome colour (visually assessed 
using a 1-9 scale) and the overall THC content of the sample. Values shown are the 
mean % w/w THC content (±SE) of all samples for each colour score. Regression line is 
shown in red. The model for this is: - Percent THC = 15.990 - 0.587 * Trichome Colour 
Score (p < 0.001 , R 2 = 0.053). 77 



XVll 



Figure 3.23. The mean CBN content (± SD) of populations of sinsemilla samples 
awarded each of the 1-9 ratings fortrichome colour. All samples were seized by police in 
2004/2005. 78 

Figure 3.24. The variability in degree of THC catabolism to CBN as related to their 
colour. Of 249 original samples 6 were rejected as outliers and not included here. 
Trichome colour was assessed visually and scored on a 1-9 scale where 1 represents 
totally clear and successively higher scores denote an increasing opacity and darkening 
in colour. 79 

Figure 3.25. Aged brown sessile glandular trichomes on 2700 year old cannabis. The 
sample is illuminated with incident light and photographed digitally. The pubescence of 
unicellular non-glandular trichomes is also clearly visible. 80 

Figure 4.1 . A smeared sample of sessile glandular trichome resin heads prepared from 
vegetative foliage of high-THC clone G1 M3. The freshly captures specimens were 
collected from the surface of a 25 urn sieve and are free-floating in water. A few pieces of 
leaf fragment are also present as a minor contaminant. 92 

Figure 5.1. Production of cannabis cuttings. A vegetative cannabis branch (a) is cut into 
sections (b). Each cutting has been cut leaving approximately five centimetre of stem 
below a single axial bud and up to one centimetre above (c). The base of the cutting is 
dipped in rooting powder (d) and then placed in moist peat plugs (e). After two weeks 
roots are protruding from the peat plugs and the cutting is ready to be planted. 111 

Figure 5.2. A newly harvested crop hung to dry on wires. 112 

Figure 5.3. An experiment to compare plant development and cannabinoid content when 
flowered in 11 and 12 hour daylengths. Plants are maintained on ebb-and-flood 
benches and lighting provided by high pressure sodium lamps. Duplicate batches of 
plants are maintained either side of the curtain with plants on the right receiving the 
longer daylength regime. 115 



Figure 5.4. A close-up view of part of an unpolllinated cannabis inflorescence, showing 
viable and older non-viable stigmas. 116 

Figure 5.5. Average monthly BRM yield (two to four crops per month) (± SD) of THC and 
CBD chemovars during the first full year of propagation. (No THC chemovar was 
harvested in April and no CBD in February and November.) 120 

Figure 5.6. The seasonal variation in cannabinoid yield of THC and CBD chemovars 
during the first full year of propagation. Values shown were estimated by combining the 
average monthly Botanical Raw Material yield (two to four crops per month) and the 
average THC or CBD content (w/w). 121 

Figure 5.7. The average yield of the THC chemovar before and after the replacement of 
mercury vapour lamps (17 W m-2) with high pressure sodium lamps (55 W m-2) of 
improved supplementary lighting (± SD). The mean is typically for four crops per month. 
No crop was harvested in April of the first year. 122 

Figure 5.8. Pattern of irradiance level in the glasshouse between 7 am and 7 pm (prior 
to the improvements in supplementary lighting) and the pattern of average monthly 
yields of THC chemovar raw material ± SD (n = 4). 1 24 

Figure 5.9. The average monthly yield as a function of the glasshouse light level at the 
beginning of flowering. On both axes the data was expressed as a percentage of the 
maximum observed (r2 = 0.92, p < 0.001). 125 

Figure 5.10. The yield of THC achieved by each of the clones (n=5) after six, eight and 
ten weeks in flower. For clarity, the clone lines have been sorted in order of descending 
THC yields after ten weeks in flower. 127 

Figure 5.1 1 . A comparison of the mean relative proportions (±SD) of THC and CBG in 
twenty five clones at three harvest dates. Analyses of variance (one-way) compared the 
proportion of THC in each clone to that in the Sativex-dependent clone G1 (shown in 
red). (* p < 0.05, **p< 0.01, ***p< 0.001). 128 



Figure 5.12. Effect of Daylength on Plant Height ± SD (n = 20) ten weeks after induction 
of flowering (* p < 0.05, *** p < 0.001 , ANOVA for individual clones and paired t-test for 
the overall mean). 133 

Figure 5.13. Effect of Daylength on Yield of Botanical Raw Material ± SD (n = 5 plants) 
ten weeks after induction of flowering (* p < 0.05, *** p < 0.001 , ANOVA). 1 34 

Figure 5.14. Effect of Daylength on Plant Height ± SD (n=20) ten weeks after induction of 
flowering. 138 

Figure 5.15. Effect of Daylength on Yield of Botanical Raw Material ± SD (n = 20 plants) 
ten weeks after induction of flowering. In the Analyses of Variance, the levels of 
significance were shown as * p < 0.05 and *** p < 0.001. 138 

Figure 5.16. Effect of Daylength on cannabinoid yield ten weeks after induction of 
flowering. (Paired t-test, two tail ** p < 0.01 ). 139 

Figure 6.1 . The mean height (± SD) of G5 M16 crop as observed at weekly intervals in 
2005. Thirty plants were measured on each occasion. Data points are shown as square 
symbols during the establishment and vegetative phase. Data points are shown as 
triangle during the flowering (generative) phase. 150 

Figure 6.2. A comparison of the pattern of stigma senescence (%, ± SD, n = 7) in 2006 
field trial plants between 10th September and 15th October with that observed in five 
consecutively grown routine indoor crops of the same variety (± SD, n = 5). 153 

Figure 6.3. Yield of Botanical Raw Material in the 2006 trial showing the effect of 
planting date and harvest date. The results are the mean dry weights (gm" 2 ± sd) 
harvested from seven replicates. 156 

Figure 6.4. Potency of Botanical Raw Material in the 2006 trial showing the effect of 
harvest date. The results are the mean % CBD (± SD) content of samples from each of 
the seven replicates, as measured by GC. Regression model, n = 7, p = 0.0028, R 2 = 
0.983. 157 



Figure 6.5. The yield of CBD in the 2006 trial, showing the effect of harvest date. The 
results are the mean CBD yields (gm" 2 ± SD) produced in each of the seven replicates. 
Regression model, p = 0.001 1 , R 2 = 0.994 157 

Figure 6.6. A comparison of the terpene profiles, as a proportion of the total peak area, of 
enriched trichome preparations prepared from glasshouse and outdoor crops (f = 
Caryophyllene Oxide). The results are the mean of five samples produced at weekly 
intervals towards the end of flowering (± SD). Glasshouse plants had been in a 12 hour 
day length for 6 to 1 weeks. Field grown crops were sampled between September 1 7 th 
and October 1 5 th . (ANOVA, Glasshouse v Field, * p < 0.05, ** p < 0.01 , *** p < 0.001 ).1 66 

Figure 6.7. Fungal damage of a cannabis inflorescence due to Botrytis cinerea. 168 

Figure 6.8. A cannabis plant at the late flowering stage. Resinous bracts are unaffected 
but leaves below the inflorescence are heavily grazed. In some cases little more than 
the midrib of the leaf remains. 169 

Figure 6.9. The rate of moisture loss of field grown cannabis, when dried at three 
temperatures (30, 40 and 50°C). The results are the mean of three crops dried in 
2004-2006 (± SD) and show the pattern of moisture loss until mean moisture content 
was < 15%. 171 



xxi 



List of Tables 

Table 1.1. Examples of plant derived drugs and modern semi-synthetic drugs made from 
the secondary metabolites of outdoor grown plants. 3 

Table 1 .2. A suggested classification of Cannabis sativa L (Sytsma et al., 2002) 8 

Table 1.3. The predominant cannabinoids found in Cannabis sativa and their main 
catabolites. 9 

Table 2.1. Photographic, microscopy and other miscellaneous items and commercial 
sources. 29 

Table 2.2. Number of each type of sample received from each constabulary. (In addition, 
one sample of cannabis powder was received from Kent). *The low number of resin 
samples from Merseyside was due to the late inclusion of such samples from this 
constabulary. 34 

Table 2.3. The median and the range of potencies of five cannabinoids (% w/w) in resin, 
herbal cannabis, sinsemilla and cannabis powder, seized in five constabularies in 
England in 2004/5. 35 

Table 3.1 The names and suppliers of the cultivars used. 50 

Table 3.2 Photographic, microscopy and other miscellaneous items and commercial 
sources. 51 

Table 3.3 The 1-9 scale for overall capitate stalked trichome resin head colour. Within 
each sample some variation would occur. 56 

Table 3.4. The potency (THC content) of yellow and green leaf tissue of the variegated 
cultivar G60-M55 assessed in each of two tests (± SD). The potency in the second test 
is also shown as a weight of THC per unit area. 81 

Table 4.1 Name, source and chemotype of clones used. 87 

Table 4.2 Miscellaneous items and commercial sources. 87 



Table 4.3. A comparison of the cannabinoid profile and content of fresh cannabis floral 
material (clone G1 M3) and sieved trichome filtrates made there from. One sample of 
each fraction was produced. Analyses show mean analytical results of four 
subsamples (±SD). Also shown is the cannabinoid content of the floral material before 
trichome extraction and the spent pulp after extraction. 90 

Table 4.4. The relative proportions of CBC and THC in sessile trichome rich preparations 
made by sieving dislodged trichomes from vegetative foliage of high-THC clone G1 M3. 

93 

Table 4.5. The proportions of principal cannabinoids (±SD) found in a trichome-rich 
filtrate clone M240, containing only sessile trichomes. One bulk sample was prepared 
and four sub-samples analysed. Also shown is the purity of the CBC (expressed as a 
%w/w of total cannabinoids detected) within the foliage from which these trichomes were 
collected. 94 

Table 4.6. The terpene profile of essential oils produced by steam distillation of glandular 
trichomes extracted from high-THC clone G2 M6 at various stages in the plant's 
development. The results are the relative peak area after analysis of the essential oil by 
GC. Missing values occur where individual terpene contents were below the detectable 
limits. 96 

Table 4.7. The terpene profile of essential oils produced by steam distillation of glandular 
trichomes extracted from high-CBD clone G5 M13 at various stages in the plants 
development. The results are the relative peak area after analysis of the essential oil by 
GC. Missing values occur where individual terpene contents were below the detectable 
limits. 97 

Table 4.8. The cannabinoid profile of the original plant material (high-THC clone G2 M6) 
from which the trichome rich preparations were made. Six subsamples were combined 
and milled to produce one sample for analysis by GC. A missing value denotes that 
cannabinoid level was below the detectable threshold. 98 



Table 4.9. The cannabinoid profile of the original plant material (high-CBD clone G5 
M13) from which the trichome rich preparations were made. Six subsamples were 
combined and milled to produce one sample for analysis by GC. A missing value 
denotes that cannabinoid level was below the detectable threshold. 99 

Table 5.1 Plant Propagation Materials 107 

Table 5.2 Germplasm Details 108 

Table 5.3 Light Measurement Equipment 109 

Table 5.4 A comparison of Botanical Raw Material yield and potency of a THC and a 
CBD chemovarwhen grown in two irradiance levels during winter. 121 

Table 5.5 The relative proportions of CBD and THC during plant development in five 
clones derived from variety G159. Results are shown as the proportion of CBD 
expressed as % of CBD+THC (± SD). The regression calculations test the significance of 
the changing proportion of CBD and THC in each clone, between the 4th and 10th week 
in 12 h daylength. 130 

Table 5.6. The falling late-summer daylength (hours. minutes) in a range northern 
hemisphere cannabis growing areas. * Contrasting locations at same latitude in 
Afghanistan, USA and Morocco. 131 

Table 5.7. A comparison of the proportion of senesced stigmas observed on ten clone 
when induced to flower in daylengths of twelve or thirteen hours. Assessments were 
made eight and ten weeks after the plants were placed in short daylength. ** Significant 
difference (p < 0.01, paired t-test). 132 

Table 5.8. Effect of Daylength on cannabinoid yield (g m-2), eight and ten weeks after 
induction of flowering. 135 

Table 5.9. The effect of day length during flowering on cannabinoid profile. Results 
shown are the proportion of CBG and THCV, expressed as a % of the CBG+THC or 
THCV+THC total, in ten clones after eight and ten weeks in short daylength. 136 



XXIV 



Table 5.10 A comparison of the proportion of senesced stigmas on ten clones in twelve 
and eleven hour daylength regimes when assessed eight and ten weeks after the 
induction of flowering. Just one overall visual assessment was made for each clone. 137 

Table 5.11 The effect of day length during flowering on cannabinoid profile. Results 
shown are the proportion of CBG expressed as a % of the CBG+THC+CBD total, in six 
clones after eight weeks in short daylength. 140 

Table 6.1. Propagation Materials and Equipment used in the field trials program to 
evaluate the outdoor propagation of Cannabis sativa L. 146 

Table 6.2. Summary of Agronomic and Yield Data from field trials performed between 
2000 and 2006. *The 2000 trial was performed before commencement of this thesis, and 
the data is included for comparison. 150 

Table 6.3. A comparison of the pattern of inflorescence development and stigma 
senescence in indoor and outdoor crops of cannabis chemovar G5. The stages of 
development of the glasshouse crop are shown from the point at which the plants are 
moved into a 12hour light/1 2hour dark until they are routinely harvested eight weeks 
later. The field crop development is shown from mid-August, just before stigma 
formation commenced. 153 

Table 6.4. A comparison of the terpene profile of freshly harvested fully mature field 
grown cannabis leaf and flower material (cultivar G5 M16) and ETP made from the same 
fresh material (2005 Field Trial). Also included is ETP made from similar mature 
glasshouse grown material. The data show the relative peak areas when assessed by 
GC at Botanix Ltd. In each case one batch of material was analysed. 155 

Table 6.5. The changing proportions of CBG and THC with respect to CBD (Mean ± sd) 
in enriched trichome preparations produced from plants harvested at weekly intervals 
between 17 th September and 15 th October. Just one ETP sample was made on each 
date after bulking together one plant from each of the seven replicates. Three 
subsamples of each preparation were analysed. 160 



Table 6.6. A comparison of the terpene profile of steam-distillates of enriched trichome 
preparations made from freshly harvested plants on five dates between 17 th September 
and 15 th October 2006. One bulked sample was analysed on each date. 163 

Table 6.7. Ratio of eight terpenes in steam distilled enriched trichome preparations made 
from freshly harvested field grown plants of cultivar G5 M16. The result for each 
terpene is expressed as a weight percentage (% w/w) of the total within each column. 
The table also shows the ratio of myrcene (the dominant monoterpene) and 
trans-caryophylene (the dominant sesquiterpene). 164 

Table 6.8. Comparison of myrcene/trans-caryophylene ratios when calculated from 
Relative Peak Areavalues and w/w data. 165 

Table 6.9. The relative proportions of THC and CBD synthesised in heterozygous BjB D 
clones derived from variety G159. The proportion of THC produced is shown as a % of 
the THC+CBD total. Just one sample of dry inflorescence material was analysed from 
each plant. 165 

Table 6.10. Summary of pest problems experienced, in decreasing order of magnitude 
using a subjective 1-5 score. * Botrytis initially minor but extremely severe in late 
harvested plots, f Symptoms not recognized as pest damage until 2005. $ Aphids 
absent in trial plots but moderate infestation of black bean aphid Aphis fabae observed 
on neighbouring cannabis seed-crop with lower secondary metabolite content. 168 

Table 6.1 1 . The level of infection with Botyrytis cinerea observed on plants harvested on 
five dates between 17 th September and 15 th October 2006. Scores are the mean % 
infection rates in the seven plots of each treatment. 169 



xxvi 



Abbreviations 



ACMD Advisory Council on the Misuse of Drugs 

ANOVA Analysis of Variance 

API Active pharmaceutical ingredient 

B D /B D Homozygous CBD chemotype 

B D /B T Heterozygous mixed THC/CBD chemotype 

BDS Botanical Drug Substance 

BMA British Medical Association 

BRM Botanical Raw Material 

Bj/Bj Homozygous THC chemotype 

cAMP cyclic Adenosine 5'- monophosphate 

CB! and CB 2 Cannabinoid Receptors 1 and 2 

CBC Cannabichromene 

CBCA Cannabichromenic acid 

CBCV Cannabichromevarin 

CBCVA Cannabichromevarinic acid 

CBD Cannabindiol 



xxvii 



CBDA Cannabidiolic acid 

CBG Cannabigerol 

CBGA Cannabigerolic Acid 

CBN Cannabinol 

CI Confidence Interval 

CNS Central Nervous System 

EMEA European Medicines Agency 

ERK Extra-cellular regulated kinase 

ETP Enriched trichome preparation 

GC Gas chromatography 

GAP Good Agricultural Practise 

GDP Guanosine 5'-diphosphate 

GMP Good Manufacturing Practice 

GTP Guanosine 5'-triphosphate 

GWP Good Wild crafting Practice 

HPLC High Performance Liquid Chromatography 

HPS High Pressure Sodium 



xxviii 



Mercury Vapour Lamp 
Metal Halide 

Medicines and Healthcare Products Regulatory Agency 
Multiple Sclerosis 

National Institute of Agricultural Botany 
Photosynthetically Active Ration 
Standard Deviation 
Standard Error 

Transmission Electron Microscope 
Tetrahydrocannabinol 
Tetrahydrocannabinolic acid 
Tetra hyd roca n na bi va ri n 
Tetrahydrocannabivarinic acid 

United Nations Childrens's Fund formally United Nations 
International Emergency Fund 

Ultra violet (Band B) 

Watts per square meter 

World Health Organisation 

xxix 



XXX 



Chapter 1 Introduction 



Chapter 1 INTRODUCTION 

1.1 Plants as a source of medicines - past and present 

Since modern man (Homo sapiens L) first walked this earth he has probably always 
exploited plants as medicines. Evidence from burial sites suggests that the 
Neanderthals (Homo neanderthalis) also shared this practice. Man's present day 
closest relative the chimpanzee (Pan troglodyte L), medicates with many plant species. 
Observations suggest that when the chimp purposefully swallows unchewed leaves of 
one of these, Aspilia mossambicensis, it is making use of the dense foliar trichomes to 
ensnare gut parasites (Huffman et al. 1996). Conceivably, common ancestors of Homo 
and Pan also shared similar practices, since many less developed animal species are 
frequently observed consuming plants for what appear to be medicinal, rather than 
nutritional, purposes (Sumner, 2000). 

There are written records dating back several millennia BCE, from early civilisations on 
most continents, which describe man's use of plants as medicines. These include 
American Indian, early European, Middle Eastern, Ayurvedic (Indian sub-continent), 
Chinese, Korean and Japanese, and Aboriginal cultures. Cannabis features in many of 
these, and in oriental and Middle Eastern countries its use can be traced back many 
thousands of years. Evidence suggests that around 3000 BCE Cannabis sativa L was 
used as an Ayurvedic (Russo, 2004) and Chinese medicine (Mechoulam, 1986). In 
Egypt, mention of medicinal uses of cannabis was written in the Papyrus Ramesseum 
III (circa 1700 BCE). More detailed uses were recorded in the Ebers Papyrus (circa 
1600 BCE) which describes the use of cannabis as a decoction in enemas, 
applications to the eye and topically in the form of medicated bandages (Mannische 
1989). An archaeological discovery of cannabis in China dating back 2700 years is 
also supportive evidence of its early medicinal use (Russo et al., 2008). In Hebrew, 
Greek and Roman texts there are references to the sedative hypnotic uses of cannabis. 
These sources refer, inter alia to its use in obstetrics and gynaecological products. 
There are also ancient references to the inhalation of smoked cannabis. Most ancient 
Middle Eastern and Asian civilisations record it being smoked for medical and ritual 
purposes. An archaeological find in Jerusalem, from the fourth century AD, indicates 
the use of cannabis vapour in an enclosed environment by women during labour (Zias 
etal., 1993). In museums around the Mediterranean (e.g. Empurius North of Barcelona) 
there are collections of surgical artefacts, which include pipes for smoking drugs. This 
is at least a millennium before the introduction of smoking tobacco from the New World. 



1 



Chapter 1 Introduction 



According to a joint UNICEF and WHO report (UNICEF, 1992) of the 80% of the 
world's population living in developing countries, only 15% had access to modern 
scientific medicine; the rest depended on traditional indigenous systems of health care 
in which herbal medicines played a part. For about three-quarters of the world 
population there is therefore complete reliance on plant-derived medicines. In 1976, in 
modern western medicine, plant derived active ingredients were still represented in up 
to 25% of prescription-drugs (Farnsworth and Morris, 1976). In developed countries 
plant-derived pharmaceuticals contribute more than $30 billion of sales revenue to the 
industry. Over 60% of anti-cancer drugs and 50% of cardiovascular and analgesic 
treatments are derived from plants (Fowler and Law, 2006). 

The World Health Organisation (WHO) reported that more than 21000 plant species 
are regularly harvested for the production of medicines. The vast majority of these are 
harvested in their natural environment. This is especially the case in areas with low 
technological and economical development (Europam, 2006). The collection of wild 
plants - so called wild crafting - has many drawbacks. Over zealous collection of wild 
plants for medicinal use has actually threatened the existence of some species e.g. 
Galanthus woronowi for the production of galanthamine, and Coleus forskohlii for the 
supply of forskolin (Evans, 2002). Consequently this type of collection has led to some 
plant species receiving CITES protection to help prevent their extinction. To maximise 
plant quality and to minimise the impact of wild-crafting on species survival and the 
environment, the modern pharmaceutical industry stipulates that the harvest should 
comply with the Good Wild-crafting Practice (GWP) Guidelines (Europam, 2006). To 
maximise the sustainable yield from some threatened wild species, research has 
established strategies for regulating and improving harvest techniques. An example is 
the Chinese tree Camptotheca acuminata (Vincent et al. 1997), which is the source of 
the major anticancer drug camptothecin, and had a market value of $5500 million in 
2006 (Fowler and Law, 2006). Wild plants are generally highly variable in their 
secondary metabolite content and they may at times also be in short supply. These 
concerns, and the threat to species survival, can at least be partly overcome by 
commercially growing crops for the pharmaceutical industry. Surprisingly perhaps, only 
about a hundred plant species are specifically cultivated for this purpose (Europam, 
2006). Growing plants for medicinal use goes back many millennia. By 660 BCE, 
Assyrian herbalists were cultivating many plants (Baker, 2002). Pliny the Elder, in his 
Naturalis Historia of 77AD, described the medicinal qualities of cannabis, and detailed 
the recommended planting and harvest timings (Pliny, 1951). 



2 



Chapter 1 Introduction 



A vast number of plant secondary metabolites form the active ingredients of modern 
drugs. By definition, these are organic compounds which are not directly involved in the 
normal growth, development or reproduction of organisms. However they add to the 
plant's survival chances by improving its resistance to predators, parasites and a range 
of environmental stresses. Some secondary metabolites are highly purified before 
being formulated as medicines. Others provide a starter material from which semi- 
synthetic drugs are produced. Examples of both types are shown in Table 1 .1 . 



Plant-derived 

ingredient/ 

precursor 


Semi-synthetic 
drug Product 


Chemical Type 


Medical Use 


Plant Source 


Codeine, 
Morphine 




Opiate alkaloid 


Analgesic 


Papaver 
somniferum 


Taxol 




Diterpene ester 


Antineoplastic 


Taxus 
Brevi folia 


Vinblastine, 
Vincristine 




Bis-indole 
alkaloid 


Antineoplastic 


Catharanthus 
roseus 


Podophyllotoxin 


Etoposide 


Lignan 


Antineoplastic 


Podophyllum 
peltatum 


Diosgenin 


Progesterone 


Aglycone 
Steroid 


Birth control 


Dioscoria 
sylvatica 


Camptothecin 


Topotecan 


Indole alkaloid 


Antineoplastic 


Camptotheca 
acuminata 


Physostigmine 


Neostigmine 


Alkaloid 


Cholinergic 


Physostigma 
venenosum 



Table 1.1 Examples of plant derived drugs and modern semi- synthetic drugs made from the 
secondary metabolites of outdoor grown plants. 

The plant kingdom has also enabled the production of so called phytopharmaceutical 
or 'botanical drugs'. These are defined as well characterised, multi-component 
standardised drugs extracted from plant sources. The medicine Veregen™, derived 
from green tea Camellia sinensis, and approved for the topical treatment of warts 
(Medigene Inc.) is such an example. In 2004 the United States Food and Drug 
Administration issued the Botanical Drug Guidance which made it possible to bring to 
market a complex mixture for which evidence of adequate safety and efficacy had been 
established (FDA, 2004). This could result in the successful company being awarded a 
period of exclusivity. By 2006 Veregen™ was the only medicine to have been 
successful (NDA 21-902). Other botanical medicines are prescribed in some European 
countries but as of 2008 none were dispensed in the UK. GW Pharmaceuticals 



3 



Chapter 1 Introduction 



commenced clinical trials in the US in 2008, to evaluate the efficacy of the cannabis- 
based botanical medicine Sativex® for the control of pain in terminal cancer patients. If 
trials were successful, a New Drug Application (NDA) would be sought. 

1.2 Cannabis Botany 

Cannabis is a tall upright annual herb. It is generally dioecious i.e. producing separate 
male and female plants (Figure 1.2a), but fibre hemp varieties have been specifically 
bred to be monoecious (hermaphrodite) (Small and Cronquist, 1976). The leaves are 
palmate, and in the iconic image of a cannabis leaf there are seven lobes, the lowest 
pair showing as backwards-facing spurs. However this number and shape is not fixed. 
On seedlings the first pair of leaves is typically monophylous (single lobed), the second 
pair having three lobes and the next pair five. In many plants, especially of central 
Asian origin, the number does not extend beyond five while in others the number can 
extend to around thirteen. Leaf size and shape differs markedly according to genetic 
origin, three contrasting examples being illustrated in Figures 1.1a - c. 




Figure 1.1. Contrasting leaf morphology in three clones of Cannabis sativa L., (a) CBD 
chemotype G5 M16 cv Gill, (b) THC chemotype Gl M3 cv Guinevere (c) Afghan landrace 
clone M146 (Illustrations by Valerie Bolas, commissioned by GW Pharmaceuticals). 

Cannabis is a wind pollinated species. The males, which are generally taller than the 
females (Figure 1.2a), commence flowering first. The specimen in Figure 1.2a was 
grown in still conditions and leaves appear yellow under the deep covering of pollen. 
When mature, the sepals on the male flowers open to expose the anthers, which hang 
freely on fine filaments (Figure 1.2c). The exposed anthers soon dehisce to shed pollen 
onto any passing air current. Shortly after the cessation of pollen production the male 
dies, but females from the same population will continue to mature for up to several 
weeks. Females produce inflorescences containing vast numbers of florets over a 



4 



Chapter 1 Introduction 



period of several weeks. This period is extended if pollen is not received. An example 
of a well-developed inflorescence is shown at the front of this thesis (Page ii). Just as 
leaf shape varies according to provenance, the shape of the inflorescence does also. 
The inflorescences of the female can become very sticky due to a covering of resinous 
glandular trichomes. These are the main source of the cannabinoids, a group of 
terpenoid compounds unique to Cannabis. Consequently, the female inflorescence is 
the most importance plant part to those exploiting it for its recreational or medicinal 
properties. Part of a fertile female inflorescence is shown in Figure 1.2b, and the fertile 
white stigmas are clearly visible against the bracts of this purple variety. 

As a result of pollination the female develops copious numbers of seeds, or more 
correctly achenes. These have been collected by man more several millennia for their 
great nutritional value, the oil produced from crushed seeds also being used for 
culinary purposes and to light lamps. The stems of some varieties are a rich source of 
fibre which has also been used by man for several millennia for the production of paper, 
rope, textiles and more latterly building materials and automotive parts (Wills, 1998). 
During the reign of Henry VIII and Elizabeth I, farmers in this country were legally 
required to grow hemp to ensure that the navy had sufficient rope and sail cloth 
(Hansard (Australia), 1996). So common was Cannabis sativa L in the English 
countryside that in his herbal of 1653 Nicholas Culpeper wrote, "This is so well known 
in this country that I shall not need to write any description of it". 




(a) (b) (c) 



Figure 1.2. (a) Male (left) and female cannabis (right) in later stage of flowering, (b) Female 
cannabis inflorescence, (c) A cluster of male flowers with sepals split open and reflexed to 
expose the anthers. 

As a consequence of its various uses, growing of Cannabis sativa L spread to all 
continents, apart perhaps from Antarctica. The original source of the species is heavily 
debated but is commonly thought to have evolved in central Asia in a region 
approximately 30° - 35°N with the Himalayas to the south, Turkestan to the west, 



5 



Chapter 1 Introduction 



Pakistan to the East and Southern China as its probable northernmost extreme (Wills, 
1998). This is just a few degrees south west of Western China where its closest relative 
the hop Humulus lupulus L is believed to have originated (Neve, 1991a). From these 
regions the two species spread and, heavily influenced by man, adapted to a range of 
latitudes, habitats and growing methods. 

1 .3 Cannabis Taxonomy 

The binomial Cannabis sativa L. carries the suffix L to record that the taxonomist Carl 
Linneaus adopted this name in his Species Plantarum of 1753. However, the binomial 
had been used much prior to this, by Leonardt Fuchs in his Kreuterbuch of 1543. Just 
as Linnaeus recognised one species, most modern day taxonomists also regard 
Cannabis as monotypic, with the species as one isolated gene pool (Harlan and de 
Wet, 1971). Within that species several subspecies are sometimes identified (Small 
and Cronquist, 1976). The biological/reproductive definition of a species states that all 
specimens of a population are of a single species if they are naturally able to sexually 
reproduce, generating fertile offspring. This is the case in the genus Cannabis, and by 
this definition therefore there are no clear biological grounds to separate it into different 
species (Schultes et al., 1974). However modern Cannabis taxonomy remains 
confused, as some scientists and commentators prefer to define species according to 
typological or morphological characteristics. Chemotaxonomy has also been used to 
catagorise cannabis populations according to their terpenoid content (Hillig and 
Mahlberg, 2004). 

In 1785 Lamark described the genus as polytypic and introduced the separate name 
Cannabis indica for plants grown in India. Such plants he regarded as being a different 
species to the European 'Cannabis sativa type' based upon their different morphology, 
geographic range, pronounced smell and greater narcotic potency. In the twentieth 
century Lamark's name Cannabis indica came to be widely used to describe the short 
wide-leaved plants indigenous to Afghanistan, like the example in Figure 1c. However, 
re-examination of the Lamark's original Cannabis indica herbarium samples shows his 
plants to have been narrow leaved. Those modern day taxonomists who adhere to a 
belief that Cannabis sativa L and Canabis indica Lam were truly separate species 
would more likely have identified Lamark's samples as Cannabis sativa L. Many other 
species of Cannabis have been proposed of which just Cannabis ruderalis Janisch has 
met wide use. This name was used to identify weak low-potency ruderal (road side) 
plants from eastern Europe which produced small marbled achenes with a strongly 
constricted abscission layer (de Meijer, 1994). 



6 



Chapter 1 Introduction 



The argument that Cannabis is polytypic gained legal significance from 1972 onwards, 
when an increasing number of court cases occurred in the USA, with defence lawyers 
challenging the taxonomy in convictions involving marijuana. United States law 
attributed the illegal recreational marijuana solely to the species Cannabis sativa L. 
Defence lawyers, claiming that their defendants were involved with Cannabis indica or 
other suggested species, argued that there was no case to answer (Small, 1976). 
Perhaps partly stemming from this challenge to the law, a large proportion of the 
commercial suppliers, advisors and commentators currently in the recreational 
cannabis industry still commonly refer to the 'species' Cannabis indica and Cannabis 
ruderalis in addition to Cannabis sativa (Snoeijer, 2002). Within this field, many 
appear to feel some empathy or romantic association with (or a perceived financial 
dependence upon) the important part that Cannabis has held within the anti- 
establishment movement. For the remainder of this thesis, the species is regarded as 
monotypic with the name Cannabis sativa L. 

Within the last hundred years the taxonomy of Cannabis above species level has also 
been cause of much debate. The genus Cannabis has most commonly been regarded 
as belonging to the family Cannabaceae of the order Urticales (Raman, 1998). 
Taxonomists originally placed Cannabis in Urticaceae (nettle family) but in the early 
20 th century some moved it to the Moraceae (fig family). However, the number of 
dissimilarities between Cannabis and the nettles or figs led to this genus being 
allocated, in the 1960s, to the new family - the Cannabaceae - along with just one 
other genus Humulus (the hops). The existence of the family Cannabaceae has gained 
widespread support. However, its place in the order Urticales (of the superorder 
Dillenidae) has been challenged in recent years. Some taxonomists now place the 
family in the order in Rosales along with the Urticaceae and the Moraceae, as shown in 
Table 1.2. 

The various suggested sub-species are described elsewhere (Raman, 1998). As 
stated earlier, chemotaxonomy has been used to split Cannabis populations between 
putative species and subspecies (Hillig and Mahlberg, 2004). European sources of 
Cannabis, usually grown for fibre or seed, typically contain a much higher cannabidiolic 
acid (CBDA) to delta-9-tetracannabidiolic acid (THCA) ratio than more tropical plants 
where the majority of the field grown illicit crops are raised. (Small and Beckstead, 
1973a, b). It has been postulated that the biosynthesis of the cannabinoids THCA or 
CBDA from the common precursor cannabigerolic acid (CBGA) is controlled by the 
presence of co-dominant alleles at a common locus. These give rise to homozygous 
chemotypes with a high THCA or CBDA purity and a heterozygous chemotype 



7 



Chapter 1 Introduction 



containing a THCA/CBDA mixture. All heterogenous cannabis populations will contain 
a mixture of all three chemotypes (de Meijer et al., 2003). Differences in the 
THCA/CBDA balance of geographically contrasting populations may be due to 
environmental pressures e.g. prevailing temperature (Boucher et al., 1974). However, 
the greatest effect on chemotype will be the selective pressure exerted man as a result 
of a local need of Cannabis for fibre, seed or drug purposes. The genotypes used in 
the research for this thesis were bred from varying putative sub-species, which 
originated from a wide range of provenances. Many of these genetic sources were 
landrace materials and others specifically-bred agricultural varieties. 



Kingdom 


Plantae - Plants 


Subkingdom 


Tracheobionta - Vascular plants 


Superdivision 


Spermatophyta - Seed plants 


Division 


Magnoliophyta - Flowering plants 


Class 


Magnoliopsida - Dicotyledons 


Subclass 


Hamamelidae 


Order 


Rosales 


Family 


Cannabaceae - Hemp family 


Genus 


Cannabis L. - hemp 


Species 


Cannabis saliva L. 



Table 1 .2 A suggested classification of Cannabis sativa L (Sytsma et al, 2002) 

1.4 UK Medicinal Cannabis Use - History and Legal Complications. 

The medicinal properties for cannabis were claimed in the UK many centuries ago. 
Interest expanded greatly in the nineteenth century following the research of British 
expatriate surgeon O'Shaughnessy, who used ethanolic tinctures of cannabis for the 
treatment of pain (Robson, 1999a). While working for the East India Company in 
Calcutta O'Shaughnessy described the methods of use of cannabis by the native 
population where It had a variety of applications, particularly as a sedative and 
analgesic. In a series of careful experiments, O'Shaughnessy defined the method of 
extraction and useful doses of the preparation so produced. In his monograph he 
described using alcohol to produce tinctures, which were increasingly imported into the 
UK. In addition to a description of the useful properties of gallenical preparations 
based on cannabis, O'Shaughnessy described some of the adverse events which 



8 



Chapter 1 Introduction 



followed its use and really established the place of cannabis tincture and cannabis 
extract in Victorian medicine. 

By the end of the century the medicinal use of cannabis was well enough established 
for it to be the subject of a reference in Merck's Manual (1899). This described 
cannabis as a hypnotic sedative and very useful for the treatment of hysteria, delirium, 
epilepsy, nervous insomnia, migraine, pain and dysmenorrhoea. General practitioners 
continued to prescribe complex cannabis formulations up until the middle of the 
twentieth century. Many of the central nervous system indications for which cannabis 
was used (analgesic, hypnotic, sedative and antiepileptic) were by then met by the 
benzodiazepine group of drugs and analgesics like paracetamol and codeine. While 
these guaranteed reliable dose control, it was difficult to obtain a consistent response 
to cannabis because of the variable cannabinoid content of the plant material available 
(Notcutt, 2004). There were also growing international concerns of social problems 
caused by recreational cannabis use. This, and awareness of the adverse effects of 
cannabis, finally led to its prohibition in the UK when the UK Government ratified the 
1925 Geneva Convention on the manufacture, sale and movement of dangerous drugs. 
It did however remain available through pharmacies, until it was completely outlawed 
as a medicine by being declared a Schedule 1 substance in the Misuse of Drugs Act 
1971. This act also outlawed the production and possession of cannabis for 
recreational purposes. Amongst three categories of decreasing seriousness described 
in Schedule 1 (A, B and C) cannabis was initially placed in Class B. This indicated that 
those caught in possession of a small or moderate quantity for personal use would 
likely attract a court fine. Repeat offenders and those supplying moderate quantities of 
cannabis would be more likely to be sentenced to a community (i.e. non-custodial 
penalty), while those producing cannabis and/or supplying large quantities could be 
imprisoned for up to fourteen years. In 2004 the UK Government reclassified cannabis 
as a Class C drug, which meant that possession could attract a much smaller 
maximum prison sentence. Possession of small quantities would be more likely to 
attract a police fine, without the offender needing to attend a Magistrates Court. In 
2009, contrary to scientific advice from the Advisory Council on Misuse of Drugs, 
cannabis was reclassified as Class B (ACMD, 2008). 

Despite its continuing prohibition, in the last twenty years in the UK, an increasing 
number of patients with severely debilitating diseases such as multiple sclerosis have 
used illicit cannabis to obtain symptom relief (Whittle, 2004). Smoking cannabis for 
recreational or medicinal reasons in the UK was almost unknown until the 1950s, 
although recreational cannabis smoking had become common a decade later (Robson, 



9 



Chapter 1 Introduction 



1999a). Most medicinal users of illicit cannabis would have also smoked the material, 
although some would have ingested it in cooked form. A number of commercially 
available books described recipes for foodstuffs containing the drug. The organized 
production of such foodstuffs for alleged medicinal use resulted in two well publicized 
convictions in 2006. A small survey carried out by the UK newspaper Disability Now in 
1997 reported that amongst two hundred medicinal users of illicit cannabis, 20% were 
using the drug to treat symptoms of multiple sclerosis and similar numbers for spinal 
injury and back pain (HLSCST, 1998). A more extensive UK survey amongst those 
using illicit cannabis medicinally received 2969 replies. It suggested that 136 diseases 
were being treated with cannabis, the most predominant being Chronic Pain 25%, 
Multiple Sclerosis 22%, Depression 22%, Arthritis 21% and Neuropathy 19% (Ware et 
al., 2005). As these numbers suggest, some respondents were using cannabis for the 
relief of more than one disease. The use of cannabis for the treatment of symptoms of 
multiple sclerosis was well known in the UK. The high incidence of usage in treatment 
of migraine and the pain of rheumatoid arthritis was perhaps less expected. In the UK 
there was relatively little use for stimulation of appetite in sero-converted patients with 
HIV and AIDS, although in the USA this was a significant indication for use of both 
smoked cannabis and the synthetic-cannabinoid product Marinol®. 

1.5 The influence of the BMA and the House on Lords Select 
Committee on Science and Technology on UK Cannabis Research. 

In 1997 the British Medical Association published a highly-influential report on the 
therapeutic uses of cannabis (BMA, 1997). The report acknowledged the weight of 
evidence for the drug's efficacy in treating spasticity, nocturia and central pain in those 
with spastic conditions such as multiple sclerosis, and stated that clinical trials in this 
field merited a high priority. The report acknowledged that cannabinoids were 
undoubtedly effective as anti-emetic agents in vomiting induced by chemotherapy and 
anti-cancer drugs. More research was recommended to identify which cannabinoids 
had the optimal therapeutic profile. The undoubted analgesic effects of some 
cannabinoids were also acknowledged. The report cited research with cannabinoids in 
cancer patients which showed that the pain control and cannabimimetic effects were 
not inseparable (Evans, 1991). The term cannabimimetic in this context is defined as 
pertaining to the pharmacological properties of A" 9 tetrahydrocannabinol, which was a 
cannabinoid identified as the main psychotropic ingredient of cannabis in 1964 (Gaoni 
and Mechoulam, 1966). Cannabinoids were envisaged as useful adjuncts to standard 
analgesics and hospices, pain control clinics and post-operative wards were suggested 



10 



Chapter 1 Introduction 



as ideal settings for such research. This option was further supported by the 
observation that mixtures of the cannabinoid THC and the opiate morphine had 
exhibited synergistic analgesic activity (Cichewicz, 2004). To facilitate trials it was 
stated that the World Health Organisation should advise the United Nations 
Commission on Narcotic Drugs to reschedule certain cannabinoids under the United 
Nations Convention on Psychotropic Substances, and in response the Home Office 
should alter the Misuse of Drugs Act accordingly. In the absence of such action from 
the WHO, the government was advised to consider changing the Misuse of Drugs Act 
to allow prescription of cannabinoids to patients. 

In the light of heightened interest in cannabis, and particularly the report by the BMA 
and the House of Lords Select Committee on Science and Technology (HLSCST) 
under the chairmanship of Lord Perry, was requested to examine the scientific and 
medical evidence to determine if there was a case for relaxing some of the existing 
restrictions on the medical uses of cannabis (HLSCST, 1998a, b). The Committee 
considered was that if there is a clinical benefit accruing from the use of cannabis or 
one of its constituents, then it should be regarded as a medicinal substance. Legally, 
under the Medicines Act, medicinal substances could only be supplied properly if they 
have been assessed by the Medicines Control Agency (now the Medicines Health 
Regulatory Agency - MHRA) and a license issued by the regulatory authority. It very 
strongly recommended that government should provide an environment in which 
interested parties could research and develop cannabis-based medicines. If their 
quality, safety and efficacy were adequately demonstrated, the developers should be 
allowed to apply for a Medicines Act License and supply the product on prescription. 

One stated criticism of an otherwise excellent report was that the BMA had focused on 
single molecules. As pointed out, herbal cannabis contains a mixture of active 
compounds, more than one of which possibly contributed to the therapeutic action. 
Sensible patients who appeared to be benefitting from herbal cannabis did not report 
an equal benefit when given just one of the active ingredients. Possible synergy 
between active compounds was a possible explanation. The action of herbal cannabis 
itself still needed definition (Wall, 1998). 

1.6 International Legal Attitudes to Medicinal Cannabis 

The United Nations Single Convention on Narcotic Drugs Schedule IV made cannabis 
the subject of special restrictions 1961. Article 2 stated that individual countries should 
protect their public using whatever means were regarded appropriate according to 
conditions prevailing in their country. Protection of the public could necessitate 



11 



Chapter 1 Introduction 



prohibition of the production, manufacture, export or import, trade in or possession of 
any such drug - except for amounts which may be necessary for medical and scientific 
research only (including clinical trails). In some countries it could be felt appropriate to 
tolerate the possession of small quantities of the drug for recreational use. 

In 1998, the UK legal regime regarding medicinal use of cannabis could be described 
as one of the most restrictive in the world (HLSCST, 1998a). The following review of 
the international legal status is not intended to be comprehensive, but describes the 
history and current situation in a number of major countries. 

1.6.1 USA 

In the early 19 th Century physicians in the USA (the world's largest pharmaceutical 
market) prescribed cannabis freely and several preparations were widely available. In 
1894 the Indian Hemp Commission Report talked favourably of cannabis drugs and 
recommended that cannabis should be controlled through taxation and regulation 
rather than prohibition. However, as in Europe, newly discovered opiate-based drugs 
were eclipsing cannabis as a medicine and its use declined. 

Through the early 20 th Century recreational cannabis use in the States was commonly 
linked with hard-drug addiction, and it came to be increasingly regarded as an 
unwelcome practise favoured by undesirable elements in society. (Bonnie and 
Whitebread, 1970). In the 1930s Harry J Anslinger, the first commissioner of the newly 
formed Federal Bureau of Narcotics, cultivated society's belief that cannabis 
threatened to destroy the country's moral fabric. One result of this was the introduction 
in 1937 of the Marijuana Tax Act, which placed such draconian burdens on those 
attempting to use cannabis for medicinal purposes that its use almost ceased. During 
the 1960s opinions in the States were beginning to change. President Nixon appointed 
The Shafer National Commission on Marijuana and Drug Abuse to review US policy 
and their subsequent report, entitled Marijuana, a Signal of Misunderstanding 1972, 
recommended a relaxation in the laws controlling cannabis use. Little changed 
however. President Nixon had declared a 'War on Drugs' and he rejected the report 
before even reading it (Russo, 2003). 

A number of patients with a firm belief in medicinal cannabis remained outspoken and 
one of these Robert Randall successfully brought a suit against the Federal 
Government seeking medically supervised access to cannabis. Citing 'Compassionate 
Use' he and others were granted legal access to 'research grade material' grown under 
government supervision in Mississippi. In 1983 thirty four States had enacted 
legislation making cancer and glaucoma patients eligible to apply for medicinal 



12 



Chapter 1 Introduction 



cannabis through this scheme. However, in 1992 the Secretary for Health and Human 
Services closed the program for new patients saying that it 'sent the wrong message' 
(Mead, 2004). Seeking to drive patients away from herbal cannabis as part of its War 
on Drugs the federal government diverted them towards synthetic THC (dronabinol). 
First formulated and launched on prescription in 1985 as Marinol the federal 
government judged that this product had rendered smoked medicinal cannabis 
unnecessary. However, absorption of this orally administered drug by the gastro- 
intestinal tract is highly variable. In contrast to smoked cannabis, patients commonly 
found it difficult to titrate the dose against their symptoms (Ohlsson et al., 1980). 

Recognising this, in 1996 the states of California and Arizona authorized seriously ill 
patients to use or cultivate, possess or use cannabis if recommended by a medical 
doctor. However, the Federal government Controlled Substances Act prohibited 
cannabis cultivation for any purpose and high-ranking Federal officials threatened that 
physicians as well as growers could face criminal prosecution. The backlash from 
doctors to this threat led the Drug Czar Barry McCaffrey to ask for review of the 
medical evidence supporting cannabis as a medicine. The report of this review 
Marijuana and Medicine: Assessing the Science Base was published in 1999 and 
included many recommendations in favour of cannabis (Joy et al., 1999). By 2008 
twelve states, covering approximately 20% of the population, authorised the use of 
cannabis for medicinal purposes. To assist these patients, a large number Cannabis 
Growers Clubs were formed. However, despite this, the federal government still 
pursued the closure of these clubs and similar outlets with vigour. In California in 2008 
over two hundred thousand patients had a written recommendation from a medical 
doctor supporting their medicinal marijuana use. Of these, 40% could be regarded as 
having a serious illness (Room et al., 2008). In this state the Federal Authorities 
appeared to be losing their battle. 

In the USA, the vehicle for regulation of research into cannabinoids is the National 
Institute for Drug Abuse (NIDA). Most of the research, which is supported in the USA, 
is directed towards mode of action studies and the cataloguing of adverse effects 
produced by cannabis. The majority of the research is concerned with preclinical 
studies and very little clinical work is supported in the USA other than investigations of 
adverse effects on the psychological profile of recreational users. The net effect of 
prohibition of cannabis in the USA has been that little or no clinical research under 
therapeutic benefit has been carried out to date. In 2009, in the last days of the Bush 
Administration, there appeared to be minimal appetite within the US government for 
any type of cannabis reform. Against this difficult background, as stated earlier, in 2008 



13 



Chapter 1 Introduction 



GW Pharmaceuticals commenced clinical trials in the US to evaluate the efficacy of the 
cannabis-based botanical medicine Sativex® for the control of pain in terminal cancer 
patients. 

1.6.2 Canada 

In 2001, Health Canada defined categories of patient eligible to receive access to 
medicinal cannabis. These included individuals with acute pain, violent nausea or 
other serious symptoms caused by multiple sclerosis, cancer, spinal injury or disease, 
AIDS/HIV, severe arthritis and epilepsy. With doctor's approval, these patients could 
receive cannabis grown under the name CanniMed by the company Prairie Plant 
Systems. It was also legal for individuals to grow Cannabis for personal medical use. 
In 2005 Sativex® received provisional approval with conditions for the treatment of 
central neuropathic pain in multiple sclerosis, and in 2007 for intractable cancer pain. 

1.6.3 Mainland Europe 

For decades, the Dutch Government has had the most relaxed attitude to cannabis use 
within Europe. Possession of small quantities of cannabis for personal recreational use 
has been tolerated. Many people used cannabis for medicinal purposes, often growing 
their own plants. Much of the cannabis used would have been bought from the large 
number of regulated 'coffee shops' that offered the sale and consumption of cannabis 
on their premises. Under the Guidance of the Ministry for Health, Welfare and Sport, in 
2003 a new source of 'medical cannabis' became available through pharmacies in the 
Netherlands. This material simply consisted of unformulated dried cannabis floral 
material. It contained high levels of THC and minimum amounts of other cannabinoids. 
The cannabinoid content was stated as being within a tightly specified range and the 
product was sterilised using y-radiation. The product was much more expensive than 
similar, albeit less-well regulated, floral material on sale in coffee shops. Perhaps 
because of this, consumption was much lower than government predictions and the 
continued supply of this material was threatened. 

In November 2006, the Ministry stated that the German and Italian governments were 
interested in accessing the Dutch medicinal cannabis. On 9 th July 2008, the Austrian 
Parliament approved Cannabis cultivation for scientific purposes under the Health 
Ministry's control. Possession of cannabis for recreational purposes remained an 
imprisonable offence. Spain has undergone a progressive decrimilisation with regard to 
cannabis possession. In 2001 the Catalonia region permitted the possession of 
cannabis for medicinal purposes and Sativex® became available in that area. 



14 



Chapter 1 Introduction 



Early in 2009 the results of a Phase III clinical trial showed that Sativex was 
significantly efficacious in treating spasticity in multiple sclerosis. This triggered an 
application for the regulatory approval for Sativex in the UK and mainland Europe. 

1.6.4 Ireland 

Cannabis is not recognised as having any medical benefits according to Irish law 
(Misuse of Drugs (Designation) Order (S.I. 69/1998). By European standards, Irish 
Courts have treated medicinal users of illicit cannabis harshly. In 2003 however, the 
Irish Medicines Board permitted clinical trials to be performed to evaluate Sativex®. 

1.6.5 Australia 

In Australia, laws differ between states. Those caught in possession of cannabis of up 
in New South Wales would only receive a fine if found with less than 15 g. Other states 
are more lenient, up to 50 g justifying a fine in Queensland, Tasmania and Victoria 
(Lenton, 2004). Cultivation of cannabis plants similarly attracts differing sentences, 
and in the Northern Territory is decriminalised. In January 2009, a four year trial 
commenced to evaluate the medical use of cannabis to treat chronically or terminally ill 
patients. 

1.6.6 Japan 

Cannabis possession in Japan is illegal for both recreational and medicinal use, and 
hefty fines and imprisonment are imposed. However, the Otsuka Pharmaceutical 
Company Ltd is collaborating with GW Pharmaceuticals Ltd to research a range of the 
cannabinoids for use in oncology and CNS ailments. The production of some of these 
cannabinoids is described in this thesis. 

1.7 The choice of active pharmaceutical ingredients (APIs) 

At the commencement of this thesis in autumn 2003 the sponsoring company 
(GW Pharmaceuticals Ltd) was propagating two feedstocks to produce the medicine 
Sativex®, which was undergoing Phase III clinical trials. One of these studies showed 
that this medicine significantly reduced spasticity symptoms in patients with multiple 
sclerosis (Wade et al., 2004). Following further studies (Barnes et al., 2006), the 
medicine became available in Canada in 2005 for the treatment of central neuropathic 
pain in multiple sclerosis and in 2007 for intractable cancer pain. These separate 
feedstocks were the dried floral and foliar material of two very different chemotypes, 
which contained predominantly A" 9 tetrahydrocannabinolic acid (THCA) or cannabidiolic 
Acid (CBDA) as the active ingredients. These terpenophenolic compounds are so 
called cannabinoids and are unique to cannabis (Turner et al., 1980). A large 



15 



Chapter 1 Introduction 



proportion of this thesis concerns the propagation, characterisation and optimization of 
these two chemotypes. At the completion of this thesis in 2009, Sativex® had received 
conditional regulatory approval for use against neuropathic pain and cancer pain in 
Canada. The medicine was also available in parts of the European Union as an 
unlicensed medicine. In addition to A" 9 tetrahydroxycannabinol and cannabidiol, 
Sativex® also contains other cannabis derived ingredients, including additional 
cannabinoids and terpenes. The acceptance of Veregen™ as a botanical medicine in 
the US opened the door to the testing there of cannabis-based Sativex®, and the 
product was subsequently approved for testing as a treatment for cancer pain, under 
the US Adopted Name (USAN) Nabiximols. 

The decision to formulate a medicine based on these two cannabinoids was due to 
several factors. Numerous in vitro and in vivo studies had shown that THC and CBD 
exhibited high levels of pharmacological activity, although the mode of action of the two 
differed markedly. (The activities of the two were compared and contrasted in a review 
by McPartland and Russo (2001)). The aim of mixing the two cannabinoids was not 
simply to draw additive benefit from the individual properties of the two molecules. 
CBD was suspected of being able to attenuate the psychoactive effects of THC, which 
were undesirable in a medicine (BMA, 1997). A growing weight of evidence also 
showed that mixtures of THC and CBD offered improved efficacy over THC-alone. 
Phytomedicines could be produced which contained both these cannabinoids, and 
these would potentially contain other additional active natural ingredients, including the 
terpenes and flavonoids (Musty, 2004). 

Due to the predilections of smokers, drinkers, tea-totallers and coffee lovers the three 
most commonly used legal drugs are nicotine, alcohol and caffeine (Robson, 1999). In 
each case the flavour of the plant-derived source is part of the enjoyment of these 
drugs. Cannabis is perhaps unique amongst illicit drugs in that, for some users, the 
wide range of tastes produced by the various sources is a similarly important part of the 
'cannabis experience' (Rosenthal, 2001). The ingredients having the greatest effects 
on the cannabis taste would most probably be the fragrant terpenes within the essential 
oils. Some of these have their own pharmacology and have been cited as likely 
synergists in mixtures with cannabinoids (McPartland and Russo, 2001). The potential 
benefit of these ingredients was demonstrated in a test measuring pain relief in mice, in 
which unknown powerful synergists produced a 330% increase in activity compared to 
THC alone (Fairbairn and Pickens, 1981). Synergistically improved efficacy of cannabis 
extracts over THC-alone was also demonstrated in a mouse model which assessed 
their antispacticity effects (Williamson, 2001). In subsequent research cannabis 



16 



Chapter 1 Introduction 



extracts also showed a significantly increased antihyperalgesic effects compared to 
CBD-alone when tested in a rat model of neuropathic pain (Comelli et al., 2008). The 
potential benefits for mankind were supported by the observation that patients taking 
synthetic derivative nabilone for neurogenic pain actually preferred cannabis herb and 
reported that it relieved not only pain but the associated depression and anxiety 
(Williamson and Evans, 2000). Reasons suggested included the more rapid absorption 
through the lung than the gut; the presence of other ingredients in plant-derived 
cannabis which might give additive or synergistic effects; and the ability of smokers to 
self-titrate their dose (Grinspoon and Bakalar, 1995). 

The decision to evaluate THC/CBD mixtures as a potential medicine was further 
supported by the knowledge that the majority of cannabis used in the UK at the time - 
at least for recreational purposes - was in the form of cannabis resin. It was suspected, 
but not confirmed, that the majority of anecdotal reports for the efficacy of cannabis in 
the UK would be based upon this material. Studies in the USA had shown that 
cannabis resin (hashish) typically contained approximately equal quantities of both 
cannabinoids, whereas herbal cannabis (marijuana) contained predominantly THC 
(ElSohly etal, 1984). 

A series of clinical trials was subsequently planned which would require the 
propagation chemotypes dominant in cannabinoids other than THC and CBD. Some of 
these are also discussed in this thesis. 

1.8 Cannabinoid and terpene biosynthesis 

The cannabinoids of greatest initial interest in this research, A" 9 tetrahydrocannabinol 
and cannabidiol are referred to as 'pentyl' cannabinoids, due the presence of a five- 
carbon chain attached to the aromatic moiety (Figure 1.3). Other pentyl cannabinoids 
frequently discussed in this thesis are cannabichromene (CBC) and cannabigerol 
(CBG). All four have important propyl analogues. The first step in the synthesis of 
pentyl cannabinoids is the condensation reaction of geranylpyrophosphate (GPP) with 
olivetolic acid. The resulting product CBGA is the direct precursor for the three major 
pentyl cannabinoids tetrahydrocannabinolic acid (THCA) (Fellermeier et al., 2001), 
cannabidiolic acid (CBDA) (Taura et al. 1996) and cannabichromenic acid (CBCA) 
(Gaoni and Mechoulam, 1966). The enzymes affecting the synthesis of these three 
cannabinoids are named THC synthase, CBD synthase and CBC synthase 
respectively. The first step in the synthesis of propyl cannabinoids is the condensation 
reaction of geranylpyrophosphate (GPP) with diverinic acid. The resultant product 



17 



Chapter 1 Introduction 



cannabigerovaric acid (CBGVA) is the direct precursor of the propyl analogues of the 
three aforementioned cannabinoids ie THCVA, CBDVA and CBCVA. 

When herbal cannabis is dried stored and heated, these cannabinoid acids 
decarboxilize gradually or completely to the neutral forms (e.g. THCA — > THC) (de 
Meijer et al. 2003). Complete decarboxilation of the acid form of the cannabinoids 
occurs during the analytical process, when using gas chromatography (Brown, 1998). 
To varying extents, and according to storage conditions, these cannabinoids undergo 
oxidative catabolism with the production of a range of additional cannabinoids. Some of 
these (eg CBN) have their own reported pharmacological activity and in some cases 
interact with other cannabinoids (Pertwee, 1998, Wilkinson et al, 2003). The principle 
active cannabinoids, their precursors and catabolites are listed in Table 1.3. 




Figure 1.3. Biosynthetic pathway of THC and THCV, via CBG or CBGV. 1, 
Geranylpyrophosphate; 2, Divarinic Acid (R1) or Olivetolic Acid (R2); 3, Cannabigerovarin 
(CBGV) (R1) or Cannabigerol (CBG) (R2);. 4, A 9 - tetrahydrocannabivarin (R1) or A 9 - 
tetrahydrocannabinol (R2). R1 (-C3H7) and R2 (-C5H11) indicate the propyl or pentyl forms of 
the metabolites; Enzyme I: geranylpyrophosphate:olivetolate geranyltransferase(GOT); 
Enzyme II: THC(V) synthase. 



18 



Chapter 1 Introduction 



Although the cannabinoids in fresh plant material exist in the acid form (THCA, CBDA 
etc.,) it is common practice to simply refer to these cannabinoids in their neutral form 
(THC, CBD etc.). In this thesis, that convention applies unless the acidity of the 
cannabinoid needs to be highlighted. 



Initial 
Biosynthetic 
Product 


Second Biosynthetic 
Product 


Decarboxylated 
Product 


Principle Initial 
Oxidative 
Catabolite 


CBGA 
Cannabigerolic 

Acid 




CBG 
Cannabigerol 






Tl 1 A 

THCA 
Tetrahydrocannabinolic 
acid 


THC 

Tetrahydrocannabinol 


CBN 
Cannabinol 




CBDA 
Cannabidiolic Acid 


CBD 
Cannabidiol 


CBS 




CBCA 
Cannabichromenic Acid 


CBC 
Cannabichromene 


CBL 

Cannabicyclol 


CBGVA 
Cannabigero- 

varic Acid 




CBGV 
Cannabigerovarin 






THCVA 
Tetrahydrocannabivarinic 
acid 


THCV 
Tetrahydro- 
cannabivarin 


CBNV 
Cannabivarin 




CBDVA 
Cannabidivanic acid 


CBDV 
Cannabidivarin 


CBSV 




CBCVA 
Cannabichromevarinic 
acid 


CBCV 
Cannabichromevarin 


CBLV 



Table 1.3 The predominant cannabinoids found in Cannabis sativa and their main catabolites. 



The ingredients in cannabis suspected of contributing to the synergistic 
pharmacological effects include the main constituents of the essential oil - the 
monoterpenes and sesquiterpenes. The precursor of cannabinoid biosynthesis GPP 
also reacts with a range of other structures to produce the monoterpenes. These are a 
very diverse chemical group, which includes cyclic and acyclic structures. They are 
relatively volatile and the main source of essence within the essential oil. In addition to 
the 'true' monoterpenes (Ci H 16 ) there are also a series of increasingly more oxidized 
families within the group. These include the alcohols (e.g. linalool C 10 H 18 O), ethers (e.g. 
1,8-cineole Ci H 18 O), esters (e.g. bornyl acetate C 12 H 2 o0 2 ), aldehydes (e.g. citral 
Ci H 16 O) and ketones (e.g. pulegone C 10 H 16 O). All of these were amongst fifty eight 



19 



Chapter 1 Introduction 



monoterpenes identified in cannabis in a detailed study by Turner et al. (1980), and 
these were accompanied by thirty eight sesquiterpenes. The latter are reported to be 
formed from a dominant precursor farnesyl pyrophosphate (FPP). Both FPP and GPP 
are derived from isopentenyl pyrophosphate (IPP). The cannabinoids, sesquiterpenes 
and monoterpenes therefore all share a common precursor in IPP. As with the 
monoterpenes, the true sesquiterpenes (C15H24) are also accompanied by series of 
more oxidized related structures. Higher molecular weight terpenes are also present, 
the next group within the 'terpenoid skeleton family' being the diterpenes. These are 
not volatile and not generally considered active constituents of an essential oil. The 
only significant example in cannabis is phytol which is a precursor and catabolite of 
chlorophyll and tocopherol Vitamin E, both of which are abundant in cannabis. Phytol 
has been shown to exhibit antispasmodic activity (Pongprayoon et al., 1992) and to 
elevate the neurotransmitter GABA levels in central nervous system (Bang et al., 2002). 
It too, may contribute to the pharmacological properties of cannabis based medicines. 

Two core pathways of IPP biosynthesis have been identified. The first of these, the 
acetate/mevanolate pathway, operates within the cytosol (the fluid component of 
cytoplasm) while the second and more recently discovered pyrovate/glyceraldehyde-3- 
phosphate pathway takes place within the specialized membrane-bound subcellular 
organelles called plastids (or more specifically leucoplasts) (Hallahan and Gray, 2000). 
These pathways are outlined in Figure 1.4. Terpene biosynthesis has been reviewed in 
detail by Croteau and Johnson (1984) and is not repeated here. 



Glucose 

I 

Triosephosphate 
▼ 

Acetyl-CoA 

I 

3-hydroxy-3-methylgluterate-CoA 

I 

Mevalonate 

I 

Isopentyl Diphosphate (IPP) 
CYTOSOL 

Figure 1.4. The two pathways of isopentyl diphosphate (IPP) biosynthesis in plants, as found in 
the plastid and cytosol respectively. 



Glucose 

I 

Glyceraldehyde 3-Phosphate (GAP) 
▼ 

Pyrovate 
GAP W 



"** C0 2 



1 -Deoxyzylulose-5-P 

I 

Isopentyl Diphosphate (IPP) 
PLASTID 



20 



Chapter 1 Introduction 



1.9 Cannabinoid Receptors and Cannabinoid Pharmacology. 

The understanding of the pharmacodynamics of the cannabinoids was greatly 
enhanced when specific cannabinoid receptors were identified in mammalian brain 
(Devane et al., 1988) and subsequently cloned (Matsuda et al., 1990). Named CBi 
receptors (Appendix 3a), these were identified in the central nervous system (CNS) 
and certain peripheral tissues. These were identified as belonging to a so-called 
superfamily of G-protein-linked receptors (Matsuda et al., 1990). G-protein-linked 
receptors characteristically exhibit a seven-folded structure which spans the cell's 
plasma membrane. In some such receptors the ligand attaches to a domain (a section 
of the polypeptide chain) that is entirely exposed on the outer surface of the cell. 
Others ligands will have domains which include parts of the chain deep within the cell 
membrane. The binding of a ligand signals any one of several activities within the cell. 
Over half of all known drugs work through G-protein receptors (Alberts et al., 2002). 
These receptors include three protein sub-units - a, (3 and y. In the unstimulated state 
of the so-called 'two state model of constitutive activity' no ligand is bound to the 
receptor 'R' and guanosine diphosphate (GDP) is bound to the a-subunit. This 
conformation the G protein is termed RGqdp- Upon stimulation, this sub-unit releases 
its GDP allowing Guanosine 5'- triphosphate (GTP) to bind in its place (Figure 1.5). 
This conformation of the G protein is termed R*G G tp(Ross, 2007). As a consequence, 
the a(3y complex dissociates into an a subunit and a (3y complex. The now-free a 
subunit is now able to change its shape and interact with target proteins. The a 
subunits have intrinsic slow hydrolase activity, and as a result the GTP spontaneously 
hydrolyses to GDP with the release of one phosphate moiety. In doing so, the G- 
protein resets itself back to the rest position. Within the cell this 'two state' model of 
constitutive activity suggests that an equilibrium exists: - 

RGgdp'* - * R*Ggtp- 

The balance of these two forms signals a series of separate reactions within the cell. 
These include alterations to the cyclic adenosine 5'- monophosphate (cAMP) 
concentration and to the activity of potassium and calcium channels. 

A- 9 THC interacts with the CBi receptors as a ligand, causing psychoactive effects 
(BMA, 1997). A second type, the so called CB 2 receptor (Appendix 3b), was 
subsequently discovered in spleen macrophages and found not to be present in the 
CNS (BMA, 1997; Pertwee, 1997). The natural function of the cannabinoid was more 
easily explained with the discovery of endogenous substances within mammalian 
tissue which interacted with these receptors. These have been termed as 



21 



Chapter 1 Introduction 



endocannabinoids to differentiate them from the plant-derived cannabinoids, which are 
sometimes called phytocannabinoids (Pate, 1994). 



receptor protein inactive G protein 




ICl 




Figure 1 .5. The disassembly of an activated G-protein into two signalling components. (Alberts 
et al, 2002) 

The first of the endocannabinoids was discovered to be the arachidonic acid derivative 
arachidonylethanolamide in 1992, and subsequently named anandamide (Devane et al. 
1992). In addition to the phytocannabinoids and endocannabinoids a number of 
synthetic cannabinoid ligands have also been manufactured. These include chemicals 
closely related to the phytocannabinoids such as the A" 9 THC analogue nabilone 
(Cesamet) and the nonclassical cannabinoid group which includes the compound 
CP 50556 (levonantradol). The pharmacological activity of many of these cannabinoids 
has been assessed in vitro and in vivo. In the former case, this has included an 
assessment of the test cannabinoid's ability to block or reverse the effects of another 



22 



Chapter 1 Introduction 



standard receptor antagonist (Pertwee, 1997). This method has been used to assess 
extracts from many of the chemotype studied for this thesis. 

The interaction of phytocannabinoids or endocannabininoids with cannabinoid 
receptors is illustrated schematically in Figure 1.6. In addition to being present in the 
central nervous system and throughout the brain, CB! receptors are also found in the 
immune cells and the gastrointestinal, reproductive, adrenal, heart, lung and bladder 
tissues. By altering the cell activity within such a wide range of tissues, several 
prominent pharmacological responses result from the agonism of CB! receptors, 
including both the control of nociceptive and neuropathic pain. CB 2 receptors are 
thought to have immunomodulatory effects and to regulate cytokine activity and 
thereby altering cell-to-cell communication. In combination with CBi receptors, they 
show pain control effects (Pertwee, 2004). 




Figure 1.6. The interaction of phytocannabinoids and/or phytocannabinoids with the CBi 
receptor in the eukaryote cell and the consequent effects on cAMP and ERK activity and ion 
channelling. Modified from Ross (2007). 

While many of the pharmacological properties of cannabis can be explained by 
interactions of its ingredients with the CB! and CB 2 receptors, not all of the effects can 
be explained this way. Cannabidiol has shown significant levels of antipsychotic 
activity, which are seen as advantageous in medicines containing A" 9 THC, but this 
cannot be totally explained by interactions with the cannabinoid receptors as CBD is a 
weak ligand (Iversen, 2008). Both CBD and A" 9 THC have been shown to have equally 
potent neuroprotective antioxidant properties in rat cortical neuron cultures, where 



23 



Chapter 1 Introduction 



glutamate otherwise reached toxic levels (Hampson et al., 1998). Indeed, the 
antioxidant abilities perhaps indicate a natural function of these cannabinoids in plant 
tissues. 

In 1998 an important trial was performed by the Royal Pharmaceutical Society of Great 
Britain, in collaboration with the UK Medical Research Council. This CAMS study 
(cannabis in MS) involved 630 patients and explored the effects of synthetic THC 
(Marinol) and a cannabis extract "Cannador" given orally on spasticity and other 
symptoms related to multiple sclerosis (Zajicek et al., 2003). The results of the study 
were mixed, and a large placebo effect was noted, but both active treatments 
demonstrated significant improvements in subjective measures of spasticity, muscle 
spasms, pain and sleep, and also in an objective measure of mobility. No effect was 
apparent on irritability, depression, tiredness, tremor or loss of energy. The authors 
noted an unexpected reduction in hospital admissions for relapse in the two active 
treatment groups. The known interaction of cannabinoids with the immune system, and 
the fact that MS was still regarded as an auto-immune condition, led them to comment 
that this finding was worthy of further investigation. 

A highly significant recent finding was the observation that p-caryophyllene, a major 
sesquiterpene in cannabis, selectively binds to the CB 2 receptor and is a CB 2 receptor 
agonist with anti-inflammatory activity in vivo (Gertsch et al., 2008). 

1.10 Aims and Outline of Thesis 

Supported by the GW Pharmaceuticals pic, this thesis was performed to improve the 
reliable production of phytopharmaceutical feedstock for the production of botanical 
medicines from Cannabis sativa L. As the title of this thesis suggests, the challenging 
research discussed here was multifaceted and covered a broad range of scientific 
disciplines. The majority of the work was divided between a pharmaceutical research 
laboratory and a glasshouse, but it also necessitated much involvement with the 
agricultural industry and law enforcement agencies. The research had five broad aims, 
as covered in Chapters Two to Six. 

The first botanical medicine from GW Pharmaceuticals to be widely tested in clinical 
trials contained the two cannabinoids THC and CBD in approximately equal quantities. 
A significant reason for choosing an equal mixture of these two cannabinoids was the 
supposition that this balance existed in most of the illicit cannabis in the UK. Chapter 
Two describes research that investigates what cannabinoid profile actually existed in 
illicit material. For many patients in the UK, such material was the only form available. 
Indeed, the anecdotal evidence that many multiple sclerosis patients were finding this 



24 



Chapter 1 Introduction 



illicit material to be efficacious was a major consideration in allowing GW 
Pharmaceuticals a license to evaluate cannabis based medicines. Despite this, little 
was known of its cannabinoid profile. As stated at the 1998 House of Lords Select 
Committee on Science and Technology enquiry into Cannabis, the action of herbal 
cannabis needed defining (Wall, 1998). The suspected 1:1 THC:CBD ratio in UK 
cannabis was based on research into the cannabinoid profile of illicit material in the 
USA. No such large scale research had been performed here in the UK. Seeking in 
part to check if a one to one ratio of the two cannabinoids was indeed the typical 
cannabinoid profile of 'street cannabis', Chapter Two describes work to evaluate the 
cannabinoid content of this material in what was the first such large scale UK study. In 
addition to supporting the business aims of GW Pharmaceuticals pic, the research also 
provided an insight into the apparently escalating potency of UK cannabis, which was 
being increasingly linked with psychotic disorders amongst Britain's youth. This data 
was eagerly welcomed by the scientific, medical and law enforcement communities. 
The co-ordination of the collection of the illicit samples from a number of constabularies 
necessitated much police time. 

The cannabinoids and most terpenes in cannabis are synthesised in small structures 
called glandular trichomes. A pharmacognosist could reasonably argue that they are 
the most important part of the plant. Chapter Three looks in depth at the differing 
structures of Cannabis trichomes, with the aim of improving the knowledge of their form 
and function. Although not all types formed cannabinoids, the inactive non-glandular 
types would frequently occur as contaminants when collecting the glandular types for 
research purposes, and they needed to be routinely identified. The aim of the work in 
Chapter Four was to compare and contrast the different glandular trichome forms and 
examine their secondary metabolite profiles. The effect of the state of trichome maturity 
on the cannabinoid content and profiles was also investigated. Techniques are 
evaluated in which trichomes are separated so as to exert some control over the 
cannabinoid profile within a phytopharmaceutical feedstock. 

From a purely botanical perspective it was essential to gain a knowledge of how the 
plant develops. From a horticultural perspective it was imperative to learn how to 
propagate plants that were healthy and high yielding. This latter aim is shared by 
growers of illicit cannabis, but the grower for the pharmaceutical industry has the 
additional aim of learning how to repeatedly propagate crops that were uniform in 
secondary metabolite content. Chapter Five describes the research to address these 
aims in an indoor environment. Chapter Six addresses the same aims but in an 
agricultural setting. Although the research is reported in sequence in Chapters Two to 



25 



Chapter 1 Introduction 



Six, the differing areas of study were performed in parallel. Growing a crop through to 
harvest takes time, especially in an outdoor environment. The horticultural and 
agricultural investigations took several years to complete. 

Addressing a seminal cannabis symposium in 1969, organised by the Institute for the 
Study of Drug Dependence, the internationally renowned expert Dr. R.E. Schultes of 
Harvard University stated that "A thorough understanding of Cannabis sativa L as a 
plant must be basic to progress in studies of its derivatives and their significance to 
man and their effects on life and social evolution" (Schultes, 1970). The author of this 
thesis very much shares that view and this motivated and influenced the studies 
reported here. 



26 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



Chapter 2 Characterisation of Illicit Cannabis in the UK 

2.1 INTRODUCTION 

It is often stated that a drug is a substance with the ability to interact with the 
metabolism of an animal (usually man). A medicine however is a beneficial drug 
formulated in such a way as to optimise its absorption and performance. The cannabis- 
based medicine Sativex® is a medicine formulated as a sub-lingual spray. In ancient 
civilisations the medicinal benefits of cannabis are achieved by smoking the dried plant 
material or resin. Much of the 'medicinal cannabis' used around the world is supplied 
as an unformulated drug. To achieve the desired effect, this material would normally be 
smoked, but it could sometimes be vaporised or ingested. 

Smoking cannabis for recreational or medicinal reasons in the UK was almost unknown 
until the 1950s, but recreational cannabis smoking had become common a decade 
later (Robson, 1999a). Most medicinal users of illicit cannabis would have also smoked 
the material, although some would have ingested it in cooked form. Organized 
production of foodstuffs containing cannabis for alleged medicinal use resulted in 
several criminal convictions in the UK in recent years. Although thought to be 
widespread, until now the true extent and pattern of medicinal use of illicit cannabis in 
the UK was not known. A number of surveys have been performed to gain a better 
knowledge of this activity. Consroe et al. (1997) asked people with MS to describe 
which of their symptoms were relieved by smoking cannabis. Spasticity and muscle 
pain were reported to show the greatest improvement (97% and 95% of responders). 
The published results did not report what form of cannabis was typically used. 

In what was thought to be the most extensive survey of illicit cannabis use for medicinal 
use among chronically ill patients, Ware et al. (2005) reported the effects of cannabis in 
2969 candidates. The patients were not selected randomly, or by any systematic 
procedure, and the study is therefore skewed towards highly motivated responders. 
82% of users smoked the cannabis and 43% reported eating it. The survey invited 
respondents' to report their preference for cannabis resin or herbal cannabis (including 
sinsemilla). The replies were not included in the published report. However, a random 
sub-sample of 500 unnamed reply forms was acquired from the authors. Just 72 of the 
500 responders had answered this question. Of these, 50 (69%) expressed a 
preference for 'herbal cannabis', 16 preferred resin (22%) and 5 (7%) were satisfied 
with both. One respondent reported a preference for 'cannabis oil'. The term 'herbal 
cannabis' in this context was used to collectively categorize materials described by 



27 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



respondents as 'herbal', 'skunk', 'bud', 'leaf, 'marijuana' and similar terms. It was plain 
from these replies that several different forms of cannabis were being used. It can be 
assumed that these would have varied greatly in cannabinoid content and profile. 
Without further research it could not be fully explained why some users preferred one 
form of cannabis over another. The British Medical Association publication Therapeutic 
uses of cannabis (1997) listed the pharmacological properties of the phytocannabinoids 
and recognised that the illicit cannabis circulating in the UK was a very inconsistent 
product and its THC content varied widely. This was confirmed by King et al. (2004) in 
the most detailed study of UK cannabis potency to date. However, the content of CBD 
and other cannabinoids in illicit UK cannabis remained little studied. 

In recent years Cannabis has been by far the most commonly used illicit recreational 
drug in the UK (Szendrei, 1997). In the early 1970s, resin consistently accounted for 
about 70% of cannabis seizures in England and Wales but from 1997 to 2002 the 
proportion fell yearly from 72% to 53% (Mwenda et al., 2005). By the following year, 
resin appeared to have been surpassed by locally grown cannabis as the main form of 
used (Hough et al., 2003). This change would be expected to affect the balance of 
THC, CBD and other cannabinoids in those cannabis materials circulating. In addition 
to having significant implications for the pharmacological properties of street cannabis, 
changes in CBD also had the potential to affect its psychoactive potential. 

It was highlighted by Smith (2005) that future studies of potency in the UK cannabis 
should include an assessment of CBD as well as THC levels. This was reiterated by 
the United Nations Office on Drugs and Crime in the World Drug Report 2006 
(UNODC, 2006). Both publications stated that CBD was a cannabinoid with 
antipsychotic activity, having the potential to alter the potential harm attributable to the 
recreational use of THC. Neither publication however was concerned how CBD might 
affect the pharmacological properties of illicit cannabis. The term 'potency' in this 
context refers entirely to the concentration of cannabinoid in a specified sample. This 
meaning of potency is widely accepted in forensic science (e.g. ElSohly et al 1984). 
However, in pharmacology potency is a measure of drug activity expressed in terms of 
the amount required to produce an effect of given intensity (Page et al 2006). In this 
chapter potency is defined as a concentration of cannabinoid. 

In the absence of a prescribable efficacious cannabis-based medicine in the UK, illicit 
cannabis continues to be used widely for medicinal purposes. However, the 
cannabinoid content and pattern of use of this material has not been comprehensively 
studied. 



28 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



2.2 AIM and OBJECTIVES 

The aim of this part of the study was to gain an understanding of the cannabinoid 
content and variability of illicit cannabis circulating in England. Within this study there 
were a number of objectives. 

2.2.7. The study would initially assess how the market was split between cannabis 
resin, herbal cannabis and sinsemilla. 

2.2.2. The cannabinoid profile of these different types would be measured and 
compared, to gain an understanding of the potential efficacy and safety of illicit 
cannabis - when used for medicinal or recreational purposes. 

2.2.3. The potency of samples would be compared to previous data, generated by the 
Forensic Science Service. The existence of any trends in THC content would 
thus be identified. 

2.3 MATERIALS 

2.3. 1 Cannabis samples 

The samples analysed from this study had been seized by police in Derbyshire, Kent, 
London Metropolitan (SE1 area), Merseyside and Sussex between 2004 and 2005. 

2.3.2 Microscopy, Photography and other Apparatus 

The microscopes and associated apparatus for this study are shown in Table 2.1 . 



Apparatus 


Source 


Photonic PL2000 - double arm cold light source. 
MX3 Low Power Light Microscope 


Brunei Microscopes 

Unit 12 Enterprise Centre, 

Bumpers Industrial Estate 
Bumpers Way, Chippenham, 
Wiltshire. SN14 6QA 


High Power Stereo Light Microscope with 
Trinocular Head for camera attachment. 


STE UK Ltd., Staplehurst Rd 
Sittingbourne, ME10 2NH 


Colour Charts 


RHS Enterprises Ltd, RHS Garden, 
Wisley, Woking, Surrey, GU23 6QB 



Table 2.1 . Photographic, microscopy and other miscellaneous items and commercial sources. 



29 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



2.4 METHODS 

2.4. 1 Collection of Representative Samples 

The collection of samples was coordinated with the assistance of the South East 
Government Home Office. Authorization was gained from the Chief Constables of 
Derbyshire, Kent, London Metropolitan, Merseyside and Sussex. This spread of 
constabularies would capture populations of differing socio-economic conditions and 
varying influences of local ports. To obtain a pool of samples which most accurately 
represented the illicit material circulating amongst cannabis users, police were 
requested to forward materials that had been seized within the last year, during street 
arrests or while raiding the property of minor drug suppliers. To help prevent any 
skewing of the data towards imported material, the study excluded large seizures from 
those arrested for smuggling cannabis into the country. Such materials would have 
likely entered the cannabis market higher up the supply chain, and not been offered 
directly to individuals purchasing cannabis for their own use. 

2.4.2 Storage of illicit cannabis samples 

Upon arrival, samples were individually numbered and stored in darkness at ambient 
temperature in 30-35% RH. All were analysed within 28 days of arrival. 

2.4.3 Categorisation of the form of each sample 

The materials were assessed visually, using a Brunei MX3 low power light microscope 
where necessary, and the form of the cannabis sample was established. Four 
categories were identified: 

2.4.3. 1 Cannabis resin 




Figure 2.1. Examples of cannabis resin samples (<lg up to 230g) seized by police in 2004/5. 



30 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



Cannabis resin consists of the glandular trichomes and other fine particles collected 
from the inflorescences and upper leaves. The material is compressed into hard 
blocks prior to importation (Raman, 1998). All samples were dark brown in colour 
(Figure 2.1). These varied in shape and size and, when present in sufficient quantity, 
generally had a light characteristic odour. 

2.4.3.2 Herbal cannabis 

As adopted by King et al. (2004), the term 'herbal cannabis' was used only to include 
imported dried plant material collected from outdoor grown plants. The material was 
light to dark brown in colour. The glandular trichomes were always brown, due to 
ageing (Mahlberg et al., 1984). Seeds were frequently present. The material was 
sometimes in loose form (Figure 2.2a), but was also frequently encountered in hard 
blocks (Figure 2.2b) where it had been compressed to reduce volume during 
importation. The material had a light fragrant odour. Fungal mycelium was 
occasionally visible, suggesting that decay had occurred at some point during 
importation or storage. 




Figure 2.2. (a) Loose herbal cannabis material showing separated seeds; (b) Compressed herbal 
cannabis material with selection of removed seeds. 

2.4.3.3 Sinsemilla 

The sinsemilla form of cannabis was light green or grey-green in colour. The material 
consisted of resinous female floral material only. Close examination often revealed 
where bracts and leaves had been physically removed. Large intact sections of 
inflorescence, up to several grams in weight, were sometimes present. More 
commonly, the material had been broken into smaller pieces and packaged into small 
packets for marketing (Figure 2.3). Seeds were always absent as a result of the all- 
female crops being grown without exposure to pollen. The glandular trichome colour 
varied between crystal clear, white and light brown. The odour was clearly stronger 



31 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



than that of resin and herbal cannabis. There was no visible sign of fungal 
deterioration. This pungent, light material was generally regarded as having been 
grown in the UK, but some could have entered the UK from mainland Europe. 




Figure 2.3. A typical confiscated sample of illicit sinsemilla cannabis consisting of three 
separate packets, each containing approximately one gram. 

2.4.3.4 Cannabis powder 

Some herbal cannabis and sinsemilla samples were recovered from portable cannabis 
grinders. These are used to break herbal cannabis and sinsemilla into a suitably fine 
texture for smoking. More complex grinders included a fine metal mesh within the 
construction, as shown in Figure 2.4b. 



Interlocking 

grinding 

components 

Sieve 

Lid 

Base and 

collected 

powder 

Resin 




(a) 



(b) 



Figure 2.4. (a) A herb grinder in closed position; (b) An open herb grinder revealing the 
component parts. 



In this example dry cannabis would be placed between the top two interlocking 
sections. As these were contra-rotated by hand, sharp projections on the face of these 
sections would abrade the cannabis and break it into small portions. These would fall 



32 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



through 4 mm diameter holes in the top right hand section onto the sieve section 
below. Any dislodged glandular resin heads would fall through this mesh into the base. 
Glandular trichomes dislodged from the plant during grinding could fall through this 
sieve and be collected in a separate chamber within the device. One grinder was found 
with approximately 1 cm" 3 of separated yellow powder. This consisted almost entirely 
of glandular trichomes. Some of these have become trapped and crushed between 
walls of the interlocking sections forming a ring of black resin. 

2.4.3.5 Other categories not included 

Previous studies on cannabis in the UK described a hash oil. This preparation is made 
by dissolving, and subsequently concentrating, cannabis extracts in an organic solvent 
(Barber etal., 1996, Hough etai., 2003). No such samples were identified during this 
study. Many samples were seized which consisted of a mixture of cannabis and 
tobacco; all were excluded. Three seized suspected-cannabis samples were also 
analysed and found to be plant material other than cannabis or tobacco. 

2.4.4 Measurement of cannabinoid potency and profile 

Where detectable the THC, CBD, CBC, CBN, CBG and THCV content of each sample 
was measured, using gas chromatography, as described in Appendix 1 . 

2.4.5 Statistical Analysis 

Analyses of variance (ANOVA), regression and F-tests were used as appropriate, 
utilising Microsoft Excel 2003 related software. Kolmogorov-Smirnov and Wilcoxon 
Rank Sum tests and Hodges-Lehman Estimates were performed using SAS software, 
with the assistance of colleagues in the Statistical Analysis Department, GW 
Pharmaceuticals Ltd. 



33 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



2.5 RESULTS and DISCUSSION 

2.5. 1 Categorisation of cannabis type between regions 

Four hundred and sixty samples were analysed in this study. The proportion of 
sinsemilla, herbal cannabis or resin in each area is shown in Table 2.1. Sinsemilla was 
the most common form found overall, accounting for 55% of the samples seized. 
However, differences were found between regions. In Kent, resin accounted for 85 of 
the 146 samples (59%), possibly due to importation through the major ports in this 
region. Herbal cannabis is presently the most common form of cannabis in the USA 
(ElSohly et al., 2000). In contrast, this study revealed this type to be the least common 
in England. Little or no herbal cannabis was identified in four of the regions, but it did 
account for 30 of the 159 samples (19%) seized in the South East London Metropolitan 
area. 



Constabulary 


Sinsemilla 


Herbal 


Resin 


Derbyshire 


15 


1 


28 


Kent 


61 





85 


London Metropolitan 


97 


30 


32 


Merseyside 


49 


1 


9* 


Sussex 


34 


3 


15 


Total 


256 


35 


169 



Table 2.2. Number of each type of sample received from each constabulary. (In addition, one 
sample of cannabis powder was received from Kent). *The low number of resin samples from 
Merseyside was due to the late inclusion of such samples from this constabulary. 



2.5.2 The range of cannabinoids in each cannabis category 

The range of cannabinoids (the cannabinoid profile) in each type of cannabis is shown 
in Table 2.2. The distribution of potencies of sinsemilla, herbal cannabis and resin 
samples were all non-parametric and the median is therefore shown as a more 
appropriate average than the arithmetic mean. The potency of resin samples varied 
widely from almost 0% THC up to nearly 11% (Table 2.2). The majority of the samples 
were at the weaker end of this range. 40% had a THC content of <2% THC, and more 
than 80% had <6% THC. The range of potencies in herbal cannabis was similar. The 
maximum THC content found was nearly 12% but approximately 90% had a content of 
<6%. 



34 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



Sinsemilla potency ranged from about 1% to 23%, the majority being toward the high 
end of this range. The cannabis powder, retrieved from the herb grinder, was the most 
potent of the samples analyzed (40.6% THC, Table 2.2). This material had been 
prepared using a very simple piece of equipment, and this illustrates that extremely 
potent cannabis preparations are readily available. 



Type 




THC 


CBD 


CBC 


THCV 


CBG 


CBN 


Sinsemilla 
(n = 256) 


Median 


13.98 


<0.10 


0.20 


<0.03 


0.41 


0.16 


Minimum 


1.15 


<0.10 


<0.10 


<0.10 


<0.10 


<0.10 


Maximum 


23.17 


0.56 


1.41 


2.74 


2.16 


2.98 


Herbal 

(n = 35) 


Median 


2.14 


<0.10 


0.22 


0.17 


0.21 


0.55 


Minimum 


0.28 


<0.10 


<0.10 


<0.10 


<0.10 


<0.10 


Maximum 


11.81 


1.97 


0.42 


0.43 


0.76 


3.62 


Resin 
(n = 169) 


Median 


3.54 


4.17 


0.34 


0.10 


0.29 


1.55 


Minimum 


0.44 


0.36 


<0.10 


<0.10 


<0.10 


0.38 


Maximum 


10.76 


6.97 


0.66 


0.29 


1.05 


4.30 


Powder 
(n = 1) 




40.63 


0.18 


0.41 


0.29 


1.59 


0.57 



Table 2.3. The median and the range of potencies of five cannabinoids (% w/w) in resin, herbal 
cannabis, sinsemilla and cannabis powder, seized in five constabularies in England in 2004/5. 



In sinsemilla samples, THC typically accounted for approximately 94% of the total 
detectable cannabinoid content. The THCV, CBG and CBN content of individual 
samples occasionally exceeded 2% w/w, but this was approximately one tenth of the 
maximum potency recorded for THC. The CBD content of sinsemilla was typically very 
low and fell below detectable levels (0.1%) in the majority of samples. The lack of the 
THC catabolite CBN, suggested that samples were comparatively fresh when seized, 
and had remained in good condition in police stores. 

THC was also the dominant cannabinoid in herbal cannabis and CBD levels were 
similarly mostly below the detectable threshold (0.1%). CBN levels were much higher 
in herbal cannabis than in sinsemilla. The ratio of THC and CBN in these samples 
varied greatly. This was at least partly due to the varying lengths of time that herbal 
cannabis encounters on its route to the UK, the majority coming overseas from South 
Africa (UNODC, 2006). A long transport period would favour the breakdown of THC to 
the catabolite CBN (Ross etal., 1997). As a result of THC catabolism, and CBD, CBG, 



35 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



CBC being more stable, THC only accounted the 65% of the median total cannabinoid 
content of herbal cannabis. 

The balance of THC and CBD in the different forms of cannabis was clearly affected by 
their contrasting genetics. Research suggests that the production of THC or CBD, from 
the common precursor CBG, is closely controlled by two co-dominant alleles at a single 
locus (de Meijer et al., 2003). As a result, cannabis plants can be identified as 
belonging to any one of three chemotypes; i.e. THC dominant, CBD dominant or an 
approximately equal mixture of the two (de Meijer et al., 2003, Small et al., 1973a). 
Sinsemilla appeared to be entirely derived from the THC dominant chemotype. Two of 
the herbal cannabis samples had a substantial CBD content due to the presence of the 
Bd gene. The resultant effect of these contrasting genetics is illustrated in Figures 2.4a 
to 2.4c. 

Cannabis resin had a very different cannabinoid profile to that of herbal cannabis and 
sinsemilla. The majority of the cannabis resin would appear to be prepared from 
landrace populations of plants which contain all three chemotypes. CBD appeared 
slightly more dominant (mean content 4.3%) than THC (mean content 3.5%) in this 
material. CBN was present in much higher quantities than in herbal cannabis or 
sinsemilla. Cannabis resin samples showed very variable contents of THC (0.4 - 
10.8 %) and CBD (0.4 - 7.0%). Compared to sinsemilla, the THC content of resin was 
significantly much more variable (two-sided f-test, p < 0.001). The ratio of these 
cannabinoids within individual samples also varied widely, as shown in Figure 2.4c. 



36 



Chapter 2 Characterisation of Illicit Cannabis in the UK 




Figure 2.5. (a) The balance of THC, CBD and CBN in sinsemilla (n = 256); (b) The balance of 
THC, CBD and CBN in herbal cannabis (n = 35); (c) The balance of THC, CBD and CBN resin 
(n = 169). 



37 



Chapter 2 Characterisation of Illicit Cannabis in the UK 

It was hypothesized that much of the variation in THC:CBD ratios within the resin 
samples was due to THC being a less stable product than CBD. Indeed, in Figure 2.4c 
resin samples exhibited a wide range of ratios between THC and its main catabolite 
CBN. To test the hypothesis, an estimate was made of the original THC content of 
each sample before catabolism to CBN had commenced. A simple arithmetic 
calculation formula was not available because the breakdown of THC to CBN is not 
quantitative (Phillips, 1998). Studies with herbal cannabis (Ross and ElSohly, 1999) 
and resin (Martone and Delia Casa, 1990) suggested that a concentration of one mole 
of CBN implied the original presence of four to six moles of THC. To estimate the likely 
correlation between the CBN content and original THC content in these samples, the 
relative contents of the two cannabinoids were plotted (Figure 2.5). 



9 i 




0.5 1 1.5 2 2.5 3 3.5 



Percent CBN 



Figure 2.6 The correlation between THC and CBN content in resin samples seized in five 
constabularies in 2004/5 (n = 169). 

The coefficient value of -2.24 suggested that the extrapolated original median THC 
content of these samples was 7.3% THC, compared to the actual median THC content 
of 3.5% at the time of analysis. Previous studies have suggested that the half-life of 
CBD in resin is approximately three times that of THC (Martone and Delia Casa, 1990). 
As a result, in the plants used to produce this resin, the original CBD content was 
estimated to have been approximately 5.5% compared to 4.2% at seizure. Figure 2.4c 
also shows that of the one hundred and sixty nine resin samples, four were almost 
devoid of CBD. These were therefore made from the THC dominant chemovar and 
probably from a very different source. 

2.5.3 Comparison of cannabis potency and profile between regions 

Sinsemilla potency ranged from about 1% to 23%, the majority being toward the high 
end of this range. There were small but statistically significant differences in mean THC 



38 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



content of sinsemilla seized in different areas. The highest mean THC content was 
found in the Derbyshire region (16.3%). This material was significantly more potent 
(p < 0.05) than that seized in London (mean 12.9%). The remaining counties returned 
mean sinsemilla potency values between these two extremes, the differences in 
potency not being statistically significant (p > 0.05). Due to the small number or 
absence of herbal cannabis samples in most regions, a meaningful comparison of 
herbal cannabis potency levels between regions was not possible. 

There were proportionally larger differences in the mean potencies of resin between 
regions. Resin seized in Sussex (6.6%) and Derbyshire (5.4%) had significantly higher 
mean THC contents (p < 0.05, ANOVA) than those seized in Kent (4.2%), London 
(3.6%) or Merseyside (2.8%). Conversely, Sussex resin was notable for having a 
significantly lower mean CBN content than that from the other counties (p < 0.05). The 
mean CBN content of the Derbyshire resin was also significantly lower than that from 
London and Merseyside. These data suggest that the Sussex and Derbyshire samples 
were less aged. 

2.5.4 Trends in Cannabis Potency 

The mean THC content of imported herbal cannabis in this study (3.1%) was lower 
than the 4.1% observed by the UK Forensic Service in 1998 (King et al., 2004). Little 
inference can be drawn regarding potency trends from this because of the small 
number of samples in both studies, the lack of a reported median in 1998 and the 
highly non-parametric data (Figure 2.7). However, the potency ranges and distribution 
patterns are broadly similar, and no major trend is apparent. 




1 1998 
]2005 



Figure 2.7. A comparison of the range and distribution patterns of THC content of seized 
imported herbal cannabis samples in 1998, King et al. (2004) (n = 44) and 2005 Potter et al. 
(2008) (n = 33). 



39 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



The mean THC content of resin (3.7%) was typical of that found previously, where 
potency levels varied from approximately 3 to 6% THC between 1 998 and 2002 (King 
et al., 2004). The mean sinsemilla potency value of 13.3% (sd 4.21%) is higher than 
that reported over the period 1995 to 2002 when the Forensic Science Service reported 
that THC content of seized cannabis rose annually from approximately 6.0 to 12.5% 
(King et al., 2004). This supports the belief that sinsemilla potency in the UK is 
potentially increasing but, due to the large standard deviation in the 2005 data, and the 
lack of reported detail in earlier data, the 2002-2005 rise cannot claimed to be 
significant. 

In both this study and in those analyses performed by the Forensic Science Service 
from 1996-98, the THC content of samples ranged from <2% THC to >20%. The range 
of sinsemilla potency levels observed in each study is compared directly in Figure 2.8. 
To assess the significance of this change in distribution of sinsemilla potencies, the 
data was analyzed using the Kolmogorov-Smirnov test for different distributions. This 
showed that the two distribution curves were significantly different (p < 0.001). A 
Wilcoxon Rank Sum test showed that the potency levels in 2005 were significantly 
higher than those in 1996/8 (p < 0.001). A Hodges-Lehman Estimate of median 
difference suggests an increase of 4.86% THC in the median potency between the 
1996/8 data and the 2005 data (C.I. = 3.77, 5.54) (Potter et al., 2008). 



25 i 




fl U <b <b N Q N 0> \k N <b N <b cfi> (ft rfr 
% u * <b' N V N t»' N <b' <fy 

% THC 



Figure 2.8. A comparison of the ranges of THC contents of Sinsemilla seized in the UK and 
analysed by the Forensic Science Service in 1996-8 (n = 145) and samples seized by police in 
Derbyshire (n=15), Kent (n=58), London Metropolitan (n = 96), Merseyside (n = 44) and 
Sussex (n = 34) in 2004/5 (total n = 247). 



40 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



Large increases in cannabis potency achieved in the 1970s were largely attributed to 
the achievements of cannabis breeders (Clarke, 2001). Many named cultivars 
produced in the 1970s and 1980s are still widely marketed. Seeds of new cannabis 
cultivars are continually produced in large numbers (Snoijer, 2002) but these do not 
appear to produce plants of significantly higher THC content (confidential GW 
Pharmaceutical data). However, cannabis seed production is unregulated and there is 
no guarantee that seeds currently marketed under established variety names are truly 
identical to those circulating thirty years previously. The rise in reported potency is 
more likely due to increasing expertise amongst the illicit UK growers in recent years, 
more of whom are able to push THC levels closer to the possible maximum. In the UK 
during this period there has been a large increase in the number of retail outlets selling 
cannabis seeds and sophisticated growing equipment. Many books, videos and DVDs 
have been produced, advising growers how to maximise cannabis potency. The 
internet has facilitated on-line purchasing of these items and has also provided easier 
access to advice on cannabis growing and processing through expert web pages and 
focused 'chat-rooms'. During this period the UK Government and Police express the 
opinion that the production and dealing of cannabis have not always been targeted 
sufficiently vigorously (Clarke, 2006). 

A recognised weakness in this study is that all the samples tested were seized by 
police from users or dealers on the street. It is not known how representative this was 
of the cannabis being consumed across the whole population. In ten years personal 
experience as a magistrate, serving both Adult and Youth Courts, a vast majority of 
offenders charged with using cannabis were seen to be from Social Grades C2 to F, as 
defined by the NRS Survey (Appendix 5), and a large proportion were young. A 
skewing in the average age of charged offenders arose because police were instructed 
to charge offenders for cannabis use if they were less than eighteen. In contrast, they 
were allowed some discretion and could issue a warning to adults. Of three million 
estimated cannabis users in England and Wales, one million were over thirty years old 
but much less likely to be charged (May et al., 2002). Research suggests that the 
decision to charge an offender, rather than issue a warning, depended partly upon the 
attitude of the offender (May et al., 2007). The more mature, circumspect and 
sophisticated user is less likely to attract police attention. The cannabis used by this 
type of user may differ in provenance, potency and price. During informal 
conversations at Multiple Sclerosis Support Group meetings, attendees frequently 
revealed that they grew their own cannabis to ensure supply, to remove contact with 
drug suppliers and to reduce costs. Hough etal. (2003) reported the same observation. 



41 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



This avoidance tactic might have significantly reduced the chances a 'medicinal' 
cannabis sample being seized. 

2.5.5 The efficacy of illicit cannabis 

The three main forms of illicit cannabis circulating in England in 2005 were very 
variable in both their potency and their cannabinoid profile. Herbal cannabis showed 
the greatest variability in THC content, this being significantly more variable than resin 
(two-sided F-test p < 0.05). Both were significantly more variable in THC content than 
sinsemilla (two-sided F-test, resin v sinsemilla, p < 0.001). Sinsemilla generally 
contained high concentrations of THC and relatively few other cannabinoids. Herbal 
cannabis was dominated by THC and CBN, the ratio of these two varying greatly 
between samples. Resin contained substantial quantities of three cannabinoids - 
THC, CBD and CBN - and the ratio of these varied greatly between samples. THC and 
CBD have been demonstrated to act together synergistically in mammalian systems 
and small differences in relative content of each could have proportionally greater 
effects on the user (Williamson, 2001). 

From its appearance, sinsemilla gave the greatest hint of its cannabinoid content. 
Although the range of THC levels in sinsemilla ranged from < 2% up to > 22%, 85% of 
samples had a THC content of between 8% and 24% THC (Figure 2.7), with the 
majority being within a closer margin of 12% to 18%. It is possible that the lowest 
potency material was also poor in appearance. Experienced users may thus have had 
an increased likelihood of being able to judge when a sample's THC content fell within 
this 12-18% band. This would have enabled a more accurate judgment to be made of 
the likely potency of the material. Being more variable in their cannabinoid profile as 
well as their potency, herbal cannabis and cannabis resin would have potentially 
demonstrated greater variations in pharmacological effect. The appearance of these 
products gave little indication of their cannabinoid content. 

Experienced users of this cannabis, who self-titrated their dose by smoking or 
vapourising, may have had the ability to adjust their intake according to the potency of 
the material. Potentially suitable blood plasma cannabinoid level could have been 
reached within a few minutes (Huestis et al., 1992). Users could thus respond quickly 
to a perceived under-dosing of cannabinoid. The variability in THC content would have 
presented greater problems for those who relied on eating cannabis-based foodstuff for 
medicinal purposes. Cannabis that is ingested would experience first-pass metabolism. 
As a result, a large proportion of the THC would be metabolised to 11-OH-THC. A 
number of other lesser metabolites would also form and plasma levels of delta 9 -THC 
would not peak until one to six hours after ingestion Consequently, those users 



42 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



ingesting cannabis would have less ability to accurately self-titrate to achieve their 
optimum dose. The final bioavailability of ingested THC is estimated to be just 6% 
compared to 10-27% during smoking (Hawksworth et al., 2004). Obviously, those who 
were supplied with low-potency cannabis may indeed find that they were unable to 
achieve sufficient THC plasma-levels, especially if ingesting the material. 

From 1994 to 1998 resin accounted for 69-72% of all cannabis seizures in England and 
Wales. From 1997 onwards the proportion of resin seizures fell annually, reaching 53% 
in 2002 (Mwenda et al., 2003). However, although resin still appeared to be the 
dominant form of illicit cannabis in the recreational market at the time, the study by 
Hough etal. (2003) and an unpublished subset of data from the patient survey by Ware 
et al. (2005), suggested that only a minority of medicinal cannabis users were choosing 
resin despite the fact that this was the way they could gain access to substantial doses 
of CBD. It has often been suggested that medicinal users appreciate the 'high' that can 
be achieved from cannabis, perhaps welcoming it as a pleasant distraction from their 
symptoms. In a published personal testimony, presented on behalf of the Alliance for 
Cannabis Therapeutics (HLSCST, 1998b), Clare Hodges stated that when treating her 
MS symptoms, she did not have to get "high" for cannabis to lift her mood and make 
her feel calm and positive. In many informal conversations with those having multiple 
sclerosis, psychoactive effects were always described as undesirable. In clinical trials 
with Sativex®, any psychoactive effects are regarded as undesirable events. There is 
therefore a paradox. Only resin contained substantial quantities of CBD, a cannabinoid 
that was efficacious in its own right and able to reduce the undesirable psychoactive 
properties of THC. In-vitro studies and limited clinical trials also suggested that 
THC/CBD mixtures were more efficacious than THC alone, when treating cancer pain 
(Johnson and Potts, 2005) and other medical conditions (confidential GW 
Pharmaceuticals data). Yet illicit users appear to have preferred to use sinsemilla or 
herbal cannabis, which evidently lacked CBD. 

The lack of preference for resin may have been due to adverse experiences 
encountered with such a variable product. It also generally contained much less THC 
than sinsemilla, and may have often been too weak to deliver sufficient active 
ingredient. For those wishing to smoke cannabis resin, the product would have to be 
mixed with tobacco or some other herbal material to support combustion. Preparation 
of such mixtures is more difficult than those incorporating herbal cannabis or 
sinsemilla. This requires a high level of dexterity which may present difficulties for 
some patients. In herbal cannabis, and more so in sinsemilla, the natural plant 
structure is still clearly visible and additives would be relatively easy to detect. In resin 



43 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



this is not possible. Large quantities of soil are reported to contaminate the material at 
harvest and adulterants added to increase yield or bind together poor quality resin 
powder (Clarke, 1998) although forensic analysis has not found clear evidence of the 
adulteration (King etal., 2004). However, suspicion remains. 

As stated earlier, many MS patients confided that they were medicating with cannabis 
that they had cultivated themselves. This finding was also reported in the small-scale 
exploratory study performed by Hough et al. (2003). Cannabis seeds of high-THC 
cannabis varieties are readily available for purchase. Screening cannabis seed 
sources, for the research described in forthcoming chapters, tests were performed to 
ascertain the chemotype of twenty four commercially available varieties. Of these, 
twenty two produced negligible amounts of CBD. Of the other two varieties, most 
seeds produced plants with a high-THC chemotype but three seedlings were of a 
heterozygous mixed THC/CBD chemotype. This approximated to 2% of all the 
seedlings tested. Patients wishing to grow their own cannabis would therefore find that, 
although seeds of a vast number of varieties are commercially available, those that 
produce CBD are rare. This suggests that, as with those buying herbal cannabis or 
sinsemilla through the illicit market, those growing their own plants are likely to be 
raising material almost devoid of CBD. 

Any medicinal value obtained from sinsemilla would be mostly attributable to the 
cannabinoid THC. However, several researchers have found that preparations made 
from the cannabis plant are more efficacious than THC alone (McPartland and Russo, 
2001, Costa et al., 2007, Ryan et al., 2007, Williamson, 2001) due to synergies 
between THC and other poorly defined components in the plant. Those active 
ingredients contributing to this synergy (such as volatile monoterpenes) may be more 
abundant in sinsemilla than in resin, thereby increasing its pharmacological activity. 
This study did not examine the content of these other agents. 

Imported herbal cannabis was typically of low potency, and large quantities of material 
would possibly be required to provide a useful medicinal effect. This may be a reason 
why some medicinal users in the UK survey reported using as much as 1 0g per day. A 
similar dosage of 7-9 grams a day of marijuana was reported to be used by those with 
chronic medicinal conditions in the USA (Conrad, 2004), where THC contents of that 
material are typically low (ElSohly, 2000). 

Research for this thesis showed that sinsemilla potency increased significantly 
between 1996 and 2005 and the efficacy may have altered accordingly. Herbal 
cannabis and resin potency appeared to have changed little. However, in recent years 



44 



Chapter 2 Characterisation of Illicit Cannabis in the UK 



the potency of cannabis products in the Netherlands has increased greatly. In 2004 
tests showed the mean THC content of resin manufactured and sold in Netherlands 
coffee shops to be 39% and CBD content just 1% (Pijlman et al., 2005). This 
compares to 4% THC and 4% CBD in resin circulating in England. Cannabis resin may 
experience similar potency trends in the UK with marked effects on medicinal efficacy. 
For those recreational users who use cannabis for the enjoyment of the psychoactive 
effects, especially the young with a disposition to psychoses, this increasingly potent 
material introduces an increased threat to safety. 

2.6 CONCLUSIONS 

As reported reported by Potter et al. (2008), cannabis circulating in England in 2004/5 
was a very unpredictable product and the THC content varied greatly. The research 
performed here recorded for the first time the variability in the CBD content and the 
THC:CBD ratios of illicit cannabis in England. This variability would affect the 
pharmacological properties of the material. A decreasing proportion of the material 
circulating was in the form of resin, which was the only significant source of the anti- 
psychotic cannabinoid CBD. The market was becoming increasingly dominated by 
sinsemilla. This was shown to have significantly increased in THC content since 1998. 

Increasing concern has been expressed in recent years, regarding the link between 
cannabis potency and psychosis - especially in teenage users (Smith, 2005). The 
implications of increasing THC content and diminishing CBD content on users of 
recreational cannabis has been well documented. For medicinal users, the variability 
in THC content also implies an unpredictability in the potential efficacy. However, to a 
certain extent the variability in THC content of sinsemilla and herbal cannabis could be 
overcome by self-titrating the dose appropriately. In resin however, the ratio of THC, 
CBD and CBN varies greatly and this variability cannot be overcome by self-titration. In 
sinsemilla and herbal cannabis, CBD is lacking and the rapid decline in availability of 
resin indicates that this cannabinoid is becoming less available to medicinal users of 
illicit cannabis. CBD is similarly scarce in commercially available cannabis varieties 
sold by seed companies, and not easily accessible to those growing their own 
cannabis. 

A powder found within a 'herb grinder' was shown to be dislodged glandular trichomes. 
This had a THC content of over 40% w/w and was thus ten times more potent than the 
average resin. This illustrated the part that glandular trichomes played in cannabinoid 
biosynthesis. It also showed how high cannabinoid purities could be achieved by 
separating these from the aerial plant material. With the ultimate aim of exploiting 



45 



Chapter 2 Characterisation of Illicit Cannabis in the UK 

cannabis trichomes, the following chapter reports research performed to gain a greater 
understanding of their form and function. 



46 



Chapter 3. Cannabis trichome form, function, and distribution 



Chapter 3 Cannabis trichome form, function, and 

distribution 

3.1 INTRODUCTION 

In Cannabis sativa L, it is widely accepted that the cannabinoids are predominantly, if 
not entirely, synthesised and sequestered in small structures called glandular 
trichomes (Mahlberg etal., 1984). Most of the monoterpenes and sesquiterpenes found 
in Cannabis were also located in these structures (Malingre et ai, 1975; Turner etal., 
1980.) Trichomes are arguably therefore the part of the Cannabis plant of greatest 
interest to the pharmacognocist. It is reasonable to suspect that the most productive 
botanical raw material would contain these glandular trichomes in substantial 
quantities, and at their optimum stage of development. Indeed, the reliable production 
of optimum feedstocks would be more likely to be achieved if the grower gained a 
greater understanding of their form, function and distribution. This would guide the 
grower how to judge trichome maturity. Similarly, improved feedstocks might be 
possible achieved by selecting plant parts with the optimum array of trichomes. As 
seen in the previous chapter, one recreational cannabis product predominantly 
consisted of trichome material. Similar enriched trichome preparations (ETPs) could 
possibly warrant evaluation as potent sources of secondary metabolites for 
pharmaceutical use. When characterising any phytopharmaceutical feedstock, a 
systematic and illustrated report of the microscopical details is generally important 
(Evans, 2002). This chapter of the thesis constitutes such a report. The following 
chapter then examines how to exploit these to full potential. 

Cannabis trichomes have been studied in depth for many years, a notable example 
being the detailed descriptive and illustrated work of Briosi and Tognini (1894), which is 
still regularly quoted. The 1970s was perhaps the period of most intensive study, much 
of the research being performed with electron microscopes and meeting the growing 
requirement for forensic identification of illicit cannabis products (Fairbairn, 1972; 
Ledbetter, 1975; Dayanandan and Kaufman, 1976; Hammond and Mahlberg, 1973, 
1977; Turner etal., 1977). 

The general term 'trichome', when applied to plants, refers to a type of epidermal 
appendage. According to one definition, 'trichomes' constitute an intermediate group of 
appendages between 'papillae' and 'emergences' (Werker, 2000). Papillae' 
(protrusions of the periclinal outer cell wall) and 'trichomes' develop from epidermal 
cells only (Uphof, 1962), whereas 'emergences' develop from both epidermal and 



47 



Chapter 3. Cannabis trichome form, function, and distribution 



subepidermal tissues (Werker, 2000). The distinction between trichomes and 
emergences is often far from distinct, and both types are commonly referred to 
collectively as trichomes (Dietmar Behnke, 1984). Plant trichomes derive their name 
from the Greek word thrix, meaning hair. They exist throughout the Plant Kingdom in 
an extremely diverse number of forms, of which over three hundred have been 
described (Payne, 1978). Their diversity has attracted much attention since the earliest 
microscopists studied them and recorded their detail (Hooke, 1665). Workers have 
chosen to catagorise trichomes in many ways according to their morphological and/or 
anatomical features. For example, Theobald et al. (1980) placed the various trichome 
morphologies under seven general headings. Dickison (1974) adopted three general 
headings: - simple, complex and glandular which were each subdivided into various 
forms. However, the major distinction was between non-glandular and glandular 
trichomes the former being differentiated by their morphology, and the latter by their 
secretory materials. 

Non-glandular trichomes, in the form of simple plant hairs, occur in the majority of 
species within the Tracheobionta (vascular plants), but glandular trichomes are found 
in just 20-30% (Dell et al., 1978). Taxonomists commonly regard the Trancheobionta 
as being divided into the Spore Forming Division, or ferns (Pteridophyta) and the 
Flowering Division (Magnoliophyta). The latter is divided into the gymnosperms 
(conifers) and the much larger angiosperms (broad leaved flowering plants). Secretory 
glandular trichomes are mostly found within the Magnoliophyta (Dell et al., 1978; Fahn, 
1988) but rarer examples are found in existing pteridophyte species, including the ferns 
Pityrogramma sp and Polypodium virginianum (Peterson and Vermeer, 1984). 
Fossilised remains of the fern Blanzeopteris praedentata reveal that glandular 
trichomes existed as long as 300 million years ago, in the Late Carboniferous period 
(Krings et al., 2003). The functions of trichomes are either guessed at or totally 
unknown and many of the hypotheses have not been experimentally tested (Werker, 
2000). These hypotheses include the deterrence of predators and protection against 
environmental stresses (Rodriguez et al., 1984; Hallahan et al., 2000; Theis and 
Lerdau, 2003; Acamovic and Brooker, 2005). Reviews by Werker (2000) and Wagner 
et al. (2004) described seventeen different trichome functions, many of which could be 
applicable in Cannabis. 

As a result of funding from the tobacco industry, the species most studied for its 
multicellular trichomes is Nicotiana tabacum (Glover et al., 2000). The plant family 
receiving most trichome studies is the Labiatae, due to the importance of the 
terpenoids to the food, cosmetic and pharmaceutical industry. This family incorporates 



48 



Chapter 3. Cannabis trichome form, function, and distribution 



peppermint {Mentha piperita), sage {Salvia officinalis), thyme {Thymus vulgaris), 
lavender {Lavendula officinalis) and over four thousand other species. Despite legal 
restrictions to plant access, cannabis trichome structures are also well studied 
(Fairbairn, 1972; Ledbetter et ai, 1975; Dayanandan et al., 1976; Hammond et al., 
1977; Mahlberg etal.,. 1984; Kim and Mahlberg 2003). There has been a great overlap 
in the knowledge gained by those studying Cannabis and other species. Cannabis 
trichome researchers have commonly described two types of non-glandular trichome, 
which have not been associated with terpenoid development. Three types of glandular 
trichome have been described on female cannabis, viz bulbous, sessile and capitate 
stalked. Males have been found to exhibit a fourth type - the antherial glandular 
trichome, which has only been found on anthers (Fairbairn, 1972). 

Photosynthesis would be the original source of the carbon utilised in the biosynthesis of 
the secondary metabolites in glandular trichomes. In some species however, e.g. 
Nicotiana tabacum, photosynthesis would actually take place in chloroplasts within the 
trichome (Akers et al., 1978). Dayanandan and Kaufman (1976) described finding 
chloroplasts within the stalks of bulbous trichomes in Cannabis (the smallest of the 
cannabis trichomes) and reported their absence in the other trichome forms. With no 
chloroplasts present, precursors of secondary metabolite biosynthesis would therefore 
have to be translocated from elsewhere. Crombie (1977) reported that cannabinoid 
biosynthesis had been observed to continue in 'sport' tissue lacking chlorophyll, 
(although data was limited and statistical analysis not possible). It would appear 
therefore that these precursors can be translocated from tissues well away from the 
trichome. 

As stated in a detailed research paper on cannabis trichomes by Dayanandan and 
Kaufman (1976), their study is essential to understand the biogenesis, distribution and 
function of the different cannabinoids. Mahlberg et al. (1984) showed that sessile and 
capitate stalked trichomes differed in their distribution, as well as their cannabinoid 
content and profile, and this was linked to the differing cannabinoid distribution in the 
plant. Recent research has greatly increased the knowledge regarding the biogenesis 
of the cannabinoids within the glandular trichomes, but further studies are required 
(Sirikantaramas et al., 2005; Taura et al., 2007). These studies have added to the 
incomplete debate regarding the function of the cannabinoids in the host plant. 

3.2 AIM AND OBJECTIVES 

In view of the central role of the trichomes in the production of cannabinoid-rich 
material the work described here investigated trichome development, structure, 



49 



Chapter 3. Cannabis trichome form, function, and distribution 



function and catabolism in Cannabis sativa L. The knowledge gained would hopefully 
be used to make recommendations as to how cannabis should be grown, harvested 
and processed to make maximum potential benefit of the secondary metabolites 
synthesised in the cannabis trichomes. Maintaining a realistic view that fellow growers 
would be unlikely to have ready access to electron microscopes, this work was 
unashamedly performed using microscopes costing no more than a few hundred 
pounds sterling. The research involved a program of studies with a range of linked 
objectives, these being: - 

3.2.1 To study and photograph the structure and apparent function of trichomes in 
Cannabis sativa L. 

3.2.2 To assess how the differing sessile and capitate stalked trichome populations 
affected the secondary metabolite content of cannabis tissues. 

3.2.3 To assess the effect of capitate stalked trichome density and colour on 
cannabinoid content and profile. 

3.2.4 To measure the effect of photosynthetic ability, or lack of ability, on the 
cannabinoid content of green and yellow tissue in variegated Cannabis sativa. 



3.3 MATERIALS 
3.3. 1 Germplasm 



Clone 


Characteristics 


Variety Name 


Supplier 


G1-M3 


High-THC 


Guinevere 


GW Pharmaceuticals 


G2-M6 


High-THC 


Galina 




G2-M7 


High-THC 


Gina 




G5-M16 


High-CBD 


Gill 




G60-M55 


Variegated 


Unnamed 




M280 


High-CBG 


Unnamed 




M186 


High-THC Pigmented 


Sweet Purple 


Paradise Seeds 

P.O. Box 377, 1000 AJ 
Amsterdam, Holland 



Table 3.1 The names and suppliers of the cultivars used. 



50 



Chapter 3. Cannabis trichome form, function, and distribution 



3.3.2 Microscopy, Tissue Stains, Photography and other Apparatus 



Apparatus 


Source 


Olympus OM10 35mm SLR camera 


Olympus UK Ltd, 2-8 Honduras St, 
London EC1Y0TX 


Olympus SP350 8 megapixel camera 
Stereo insert 30mm lens tube, 
SP-350 Unilink (universal adaptor) 


Brunei Microscopes 

Unit 12 Enterprise Centre, 

Bumpers Industrial Estate 
Bumpers Way, Chippenham, 
WILTS. SN14 6QA 


Phntnnio PI 9000 Hniihlo arm onlrl linht 
rllUlUMIU r LlUUU UUUUIc? d 1 1 1 1 UUIU nyi'i 

source. 


ivi/\o i_uw ruwci i_!yiiL ivml-i uoL-upt- 


I— linh Pnwor Qtoron I inht l\/li/"*i*ncr , nr\o \A/ith 

Trinocular Head for camera attachment. 


o I C Ur\ LIU., OlapicMUIol nU 

Sittingbourne, ME10 2NH 


Eye Piece Graticule for Specimen Size 
Measurement 


Hemp Oil (Culinary grade) for mounting of 
microscope specimens 


Motherhemp, Springdale Farm, 
Rudston, East Yorkshire Y025 4DS 


RS 732-139 Hole Punch Kit 


RS Components Ltd., Birchington Road, 
Corby, Northants, NN17 9RS, 


Fast Blue Tissue Stain 
2,3,5-Tetrazolium Chloride Stain. 
Glycerol 


Sigma-Aldrich Company Ltd., Fancy 
Road, Poole, Dorset. BH12 40 H 



Table 3.2 Photographic, microscopy and other miscellaneous items and commercial sources. 



3.4 METHODS 

3.4. 1 Photomicrograph Studies 

3.4.1.1 Choice of Microscopes 

Two levels of light microscopy were used in this study. For more general observations 
of plant tissue a low power microscope was employed. Using a Brunei MX3 binocular 
microscope, fitted with WF10X eyepieces and 1X or 3X objectives, samples of plant 



51 



Chapter 3. Cannabis trichome form, function, and distribution 



tissue up to 15 mm in diameter were observed. For more detailed observations, of 
small numbers or individual trichomes, a higher level of magnification was required. 
For this, a high power stereo light microscope fitted with x10 eyepieces and x4, x10 
and x40 objectives was utilized. This gave fields of view of up to 4.5mm in diameter. 

3.4.1.2 Staining 

Two stains were utilised to aid visibility of trichome internal cell strictures and to gain 
further clarification of the sites of secondary metabolite biosynthesis and storage. The 
first stain fast blue, which is commonly used when analysing cannabinoids by thin layer 
chromatography, has also been used during histochemical studies on cannabis 
trichomes (Andre and Vercruysse, 1976). A 0.3% w/v aqueous solution stains 
cannabinoids orange or pink, but other phenolic compounds are also stained. 

The second stain used was the 'vital stain' 2,3,5-Triphenyl tetrazolium chloride, also 
known as TTC or tetrazolium red. This is an almost colourless water-soluble stain. In 
the presence of viable tissue this is reduced (probably by dehydrogenase enzyme 
activity) to insoluble red triphenyl formazan (Smith, 1951). A 1% w/w solution was 
used for rapid staining (< 1 hour) and a weaker 0.1% w/w used when slowly staining 
samples overnight. 

3.4.1.3 Unmounted Sample Preparation 

The majority of the low power microscope observations were made on unmounted 
specimens. For these, small pieces of plant tissue were cut from the plant and placed 
directly onto the low-power microscope plate. As a result, areas of pubescence 
containing over one hundred trichomes could be observed in a single view. The same 
minimalist method of sample preparation was sometimes utilised when viewing plant 
tissue on the high power microscope. However, to achieve views where large 
proportions of the material were simultaneously in focus, flat samples were likely to 
produce success. For this, areas of tissue up to a few millimetres in diameter would be 
laid flat on a microscope glass slide. Trichome filtrates were typically observed by 
smearing these onto a glass slide. 

3.4.1.4 Mounted sample preparation 

All the mounted samples prepared in this study were designed to be temporary, and 
disposed of immediately after use. To mount intact pieces of plant tissue, small pieces 
up to 1cm diameter were placed on a glass slide and a few drops of mounting fluid 
placed on the specimen. A cover slip was placed on an angle above the specimen, 



52 



Chapter 3. Cannabis trichome form, function, and distribution 



with one edge of the slip touching the slide. The cover slip was then lowered onto the 
specimen. As a result, excess fluid and trapped air bubbles would be expelled from the 
underside of the slip, and removed with paper tissue. 

Water (refractive index 1.33) was sometimes used as a mounting medium. However, 
transparency of biological samples is best achieved by selecting a medium with a 
refractive index closest to that of the subject (Delly, 1988). Conversely, definition of 
colourless objects is increased by choice of a mountant with a refractive index different 
from that of the object, the ideal ratio for clarity being 1.06 to 1.00 (Evans, 2002). The 
refractive index of glandular trichome contents was not known. However, initially 
aiming for transparency and knowing the refractive index of two of the major 
constituents of glandular resin head - myrcene and trans-caryophyllene - to be 1 .48 
and 1 .50, hempseed oil and glycerol (refractive index 1 .47) were selected. 

3.4.1.5 Illumination 

The Brunei MX3 low power microscope incorporated two light sources. These could be 
directed vertically upwards and/or downwards onto the subject. The material was 
sometimes illuminated, by incident light, using a Photonic PL2000 - double arm 'cold 
light source'. By the inclusion of optical fibres, this enabled white light from a halogen 
lamp to be directed at the subject without any accompanying radiant heat. Alteration of 
the flexible illuminating arms enabled the angle of incidence of light to be adjusted. 

Some samples, when placed on the STE high power microscope, were also illuminated 
using the cold light source while others were illuminated from below. When viewing 
samples mounted beneath a cover slip, the microscope was set up using the Kohler 
illumination method (Delly, 1988), which first ensures that the light from the condenser 
lens is correctly focused on an empty microscope slide. When viewing unmounted 
specimens the condenser height and aperture were adjusted while viewing the subject 
until optimum resolution was achieved. 

3.4.1.6 Photography 

To enable photographs to be taken through the low power microscope, one eyepiece 
was replaced with a compatible 30 mm lens tube to which an Olympus OM10 35 mm 
single lens reflex camera or Olympus SP350 8 megapixel digital cameras would be 
attached. As in ordinary photography, the depth of field is considered to be the distance 
from the nearest object plain to the farthest object plain that is in focus. When objects 
are a long distance from the camera lens the depth of field is large. However, depth 



53 



Chapter 3. Cannabis trichome form, function, and distribution 



decreases as the image comes closer to the lens. When taking photomicrographs, 
depth of field is measured in micrometers (Delly, 1988). To maximize the chance of 
finding substantial areas of tissue simultaneously in focus within this narrow depth of 
field, multiple samples were laid as flat as possible on glass slides. Aided by surface 
tension, samples mounted beneath a cover-slip were more likely to be retained within a 
narrow area of focus. 

The earlier studies in this research used single lens reflex photography and colour print 
film. For high magnification situations, the required exposure times occasionally 
exceeded ten seconds. Following brief tests (data not shown) high-speed ASA 400 
film was selected. This reduced exposure times while maintaining sufficiently high 
resolving power. Later studies were performed using digital photography. In all cases, 
photomicrographs were taken on a solid bench and the shutter activated remotely to 
reduce manually-induced camera-shake. 

3.4. 1.7 Isolation and Observation of Detached Glandular Resin Heads 

Adapting a method developed for studying trichome resin heads of thyme Thymus spp, 
(Yamaura, 1992) individual resin heads from capitate stalked trichomes were 
simultaneously removed and fixed on adhesive tape. One layer of sellotape® was 
tightly wrapped around the handles of a domestic clothes peg with the adhesive 
surface facing outwards. The taught adhesive surface was then allowed to touch the 
surface of cannabis bracts where glandular stalked trichomes were present. The 
adhesive surface was promptly fixed to a clean microscope slide. Detached glandular 
heads were occasionally trapped in the adhesive and readily viewed. 

3.4.2 Effect of glandular trichome array on the secondary metabolite 
content of plant tissues 

For this study, bracts of nearly-mature cannabis inflorescences were selected where 
the pubescence of capitate stalked trichomes was only visible on approximately half of 
the area (Figure 3.1). In such cases, it was always the proximal tissue that displayed 
this pubescence. Twenty bracts of each of three high-THC clones were selected. 
These were cut with a scalpel to separate each bract into portions where stalked 
trichomes were judged to be present or absent. Using a low power dissection 
microscope, the density of stalked and sessile trichomes in the separate tissues was 
assessed by counting the number, within a single randomly-selected field of view of 
16.6 mm" 2 , on both the upper and lower surface of each of these bract sections. 



54 



Chapter 3. Cannabis trichome form, function, and distribution 



Samples were then bulked to produce one sample of proximal bract tissue, and one 
sample of distal tissue, for each of the three clones. The samples of proximal and distal 
bract material of were then separately dried in an oven at 40 e C for twenty four hours. 
The cannabinoid content was assessed by GC (Appendix 1). 



Female Flowers 



Distal region of bract devoid 
of capitate stalked trichomes 



Proximal region, densely 
covered in a pubescence of 
capitate stalked trichomes 




Figure 3.1. Upper surface of a bract within a cannabis inflorescence showing glandular stalked 
trichomes to be present only within the proximal region (Potter, D.J.). 

3.4.3 Organoleptic assessment of the effect of trichome colour and 
pubescence density on cannabis potency. 

This study utilised over two hundred and fifty illicit samples of sinsemilla cannabis 
inflorescence seized by police from five constabularies during 2004/2005 (Potter et at., 
2008). These were collected from police storage and relocated to a central 
dehumidified store (30% +/- 5% RH) and kept in darkness at ambient temperatures 
prior to assessment. 

All samples were visually examined using a Brunei MX3 low power microscope. 
Trichome density and trichome colour were awarded single overall scores on a 
subjective 1-9 scale (Table 3.3). This was based upon those commonly used by the 
National Institute of Agricultural Botany for the subjective assessment of plant 
characteristics (NIAB, 2007). For trichome density a score of 1 was awarded to the 
maximum trichome density and progressively higher scores denoted a thinner 
pubescence with a score 9 denoting that no intact glandular stalked trichomes were 
visible. For trichome colour a score of 1 denoted a sample within which the vast 
majority of glandular trichome heads were completely clear. With maturation these 
resin heads can typically become turbid and then brown. The score would be awarded 



55 



Chapter 3. Cannabis trichome form, function, and distribution 



for the colour of the majority within the sample. The samples were subsequently 
analysed for cannabinoid content by GC (Appendix 1). 



Score 


Density 


Colour 


1 


Maximum Density 


Clear 


2 


Extremely Dense 


Misty White 


3 


Very Dense 


Translucent 


4 


Moderately Dense 


Opaque White 


5 


Intermediate Density 


Slightly Brown 


6 


Scarce 


Light Brown 


7 


Very Scarce 


Mid Brown 


8 


Extremely Scarce 


Dark Brown 


9 


Absent 


Very Dark 



Table 3.3 The 1-9 scales for overall capitate stalked trichome density and resin head colour. 
Within each sample some variation would occur. 

3.4.4 Effect of photosynthetic ability, or lack of ability, on cannabinoid 
biosynthesis in sessile trichomes. 

This study utilised a very rare variegated cultivar of Cannabis sativa L, G60 M55. In a 
first of three tests, three plants were selected and leaves collected where pure yellow 
and dark green could be viewed on either side of the leaf's midrib. Using dissection 
scissors, areas of approximately equal size were sampled from symmetrically opposite 
areas on each side of the leaf's midrib. These varied in size but were typically each no 
more than 100 mm 2 . For each colour tissue, one bulked sample was produced per 
plant - each containing twenty portions of leaf. The emphasis on symmetry was 
designed to ensure that the green and yellow samples were at identical stages of cell 
expansion. Samples were oven-dried at 40 e C for 24 hours and analysed for 
cannabinoid content by GC (Appendix 1). In a second test the above procedure was 
repeated with just two plants. 

A third test was performed where the cannabinoid quantification was based upon leaf 
area rather than leaf weight. For this test a disk cutter was used to cut equal sized 
disks from totally green or yellow areas. Leaves of even shape were selected where 
disks could be cut from symmetrically-located pure yellow and green areas (Figure 
3.2). Twenty disks (1 cm 2 diameter) of each colour were cut from each of two plants. 



56 



Chapter 3. Cannabis trichome form, function, and distribution 



Samples were dried as in the above test. From each plant, the twenty disks of each 
colour were bulked, thereby providing two replicates per colour. These were analysed 
by GC. 




Figure 3.2. A variegated leaf of clone M60, with 1cm diameter disks cut from symmetrically 
opposite sides of the midrib (Potter, D.J.). 

In both tests the samples were viewed through a microscope and an assessment of 
sessile trichome density was attempted. 

3.4.5 Statistical Methods 

Basic calculations of SD (standard deviation), SE (standard error) and ANOVA 
(analyses of variance) and regression were performed using Microsoft Excel software. 

3.5 RESULTS AND DISCUSSION 
3.5. 1 Photomicrograph studies 

All six forms of trichome described by Fairbairn (1972) were examined. These are 
described in turn. 

3.5.1.1 Simple unicellular trichomes 

An example of this type is shown in Figure 3.3a. The trichome is seen to have 
developed from a cell within the epidermis. These simple trichomes, also known as 
covering trichomes, were the first to appear. These were initially observed on the 
surface of cotyledons immediately after germination. This form continued to develop in 
abundance on the underside of leaves (and to a much lesser extent on the upper 



57 



Chapter 3. Cannabis trichome form, function, and distribution 



surface) throughout the plant's life. These trichomes were typically orientated to face 
towards the distal part of the leaf, bract or bracteole and in many instances would lie 
almost flat on the surface. This pubescence of trichomes would cover the underside of 
the leaf with a layer of trapped air, thereby reducing water loss and providing some 
insulation against extreme temperatures (Rodriguez etal., 1984). 

3.5.1.2 Cystolythic trichomes 

This type of trichome is shown in Figure 3.3b. Specimens were first observed on the 
upper surface of the initial pair of true leaves on a cannabis seedling. Always pointing 
towards the distal part of the leaf, these trichomes gave the upper surface a texture 
that was rough to the touch. 

At the base of each trichome is a cystolyth. These concretions are common throughout 
the plant kingdom and are typically formed from calcium oxalate or calcium carbonate 
crystals. Those found in Cannabis are of the latter type (Dayanandan and Kaufman, 
1976; Evans, 2002). These tough trichomes would presumably reduce the palatability 
of the foliage to leaf-eating predators. Histochemical chemical staining of these 
trichomes with fast blue occasionally resulted in pigmentation of these organelles, but 
the presence of phenolic substances on these trichomes was attributed to 
contamination from leaking capitate stalked trichomes. This supposition was supported 
when the vital stain tetrazolium red was used. No reduction of tetrazolium was 
observed apart from in the cells immediately surrounding the cystolyth, where 
respirative activities accompanied the formation of these concretions. 



Figure 3.3. (a) Unicellular non-glandular trichome. The sample is temporarily mounted under 
hemp oil and viewed in transmitted light; (b) Cystolythic trichomes observed on the leaf margin 
of a young leaf. The sample was temporarily dry-mounted and viewed in transmitted light. 
Cystolyths (concretions of calcium carbonate) are visible at the base of each trichome 
(Potter, D.J.). 




(a) 



(b) 



58 



Chapter 3. Cannabis trichome form, function, and distribution 



3.5. 1.3 Capitate sessile trichomes (more commonly simply called sessile trichomes) 



Figure 3.4. (a) a capitate sessile trichome observed on the edge of one of the first pair of true 
leaves of a cannabis seedling. The specimen was temporarily dry-mounted and viewed using 
both transmitted and incident light; (b) a sessile trichome on a leaf surface stained with Fast 
Blue. The still-wet sample was temporarily dry-mounted and viewed using incident light 
(Potter, D.J.). 

Apart from on the cotyledons and the supporting hypocotyl, sessile trichomes were 
observed on all other aerial surfaces throughout the plant's lifespan. The example 
shown in Figure 3.4a was a very rare find, being situated on the margin of one of the 
first pair of monofilous leaves of a cannabis seedling. This location enabled the 
specimen to be observed in profile, without the need of microtome sectioning. 

Those previously studying cannabis trichomes have referred to this type as sessile 
(Latin sessilis- sitting) or capitate sessile (Latin caput - head). By definition this type 
does not have a stalk. The trichome is in fact connected to the mesophyl cells via a 
stalk cell, but this is hidden beneath the trichome resin head. The stalk cell was seen to 
contain chloroplasts, enabling some photosynthetic activity. This form of trichome is 
found in many other plant families and, due to its flattened shape and short stalk, is 
often referred to as a 'peltate' trichome (Latin pelta - a short-handled hand-held round 
shield). According to some plant physiologists, the development of this structure from 
sub-epidermal cells defines this structure as an 'emergence' rather than a 'trichome' - 
the latter always developing from an epidermal cell. However, as stated earlier, most 
authors regard both structures as 'trichomes' (Werker, 2000). 

The glandular head (or resin head) incorporates a disc of secretory cells at the base. 
These appear to totally lack chlorophyll. Above the secretory cells, and below the 
trichome's outer membrane, is a chamber within which the secretory cells sequester a 
resinous mixture that includes cannabinoids and essential oils (Mahlberg et al., 1984). 




(a) 



(b) 



59 



Chapter 3. Cannabis trichome form, function, and distribution 



Mature sessile trichomes are reported to typically have eight secretory cells within the 
disk. When the trichomes were viewed from the side, as in Figure 3.4a, it was not 
possible to confirm this. When illuminated and viewed from overhead the disk of 
secretory cells was also difficult to observe. This problem was exacerbated by the fact 
that the glandular head itself acted as a powerful convex lens. As a result it was difficult 
to gain an undistorted view of structures with the glandular head. As the sessile 
trichomes matured, the interior of the structure would typically turn translucent or 
opaque white, further hampering observations of internal structure. Trichomes would 
sometimes turn brown in very mature or aged samples. A clearer confirmation of 
secretory cell numbers was achieved by immersing the cannabis leaf in fast blue stain 
for ten minutes and then rinsing in water immediately prior to examination. Trichomes 
varied in the speed at which the stain was absorbed and assimilated. Some samples 
exhibited very little staining while others became totally red, allowing no visible 
differentiation of tissues. In ideal situations only the membranes of the secretory cells 
were stained, allowing a clearer view of the trichome's internal structure (Figure 3.4b). 
Their function is not known but across the Plant Kingdom their role is guessed to be the 
protection of the plant tissue against predators. 

3.5.1.4 Antherial Sessile Trichomes 

Sessile trichomes were also observed on cannabis anthers. The observations 
supported the theory of Fairbairn (1972) that these 'antherial sessile trichomes' were a 
distinct form of trichome. These antherial trichomes were different from all other 
sessile trichomes by virtue of their larger size (Figure 3.5). With a diameter of 
approximately 70-80|im these unique sessile trichomes were significantly larger than 
those located elsewhere. Sessile trichomes were also generally present on the calyx 
surrounding these anthers, but these were of the more normal smaller form. Very 
similar antherial trichomes are observed in the furrows of the anthers of the male hop 
Humulus lupulus L. (Neve, 1991a). 



60 



Chapter 3. Cannabis trichome form, function, and distribution 




(a) (b) 

Figure 3.5. (a) a row of antherial sessile trichomes showing their normal distribution in the 
furrow of a cannabis anther. These anthers were captured in incident light through a low-power 
microscope, (b) closer view of antherial trichomes (Potter, D.J.). 



3.5.1.5 Capitate Stalked Trichomes 

These trichomes were generally abundant on the calyx, bracteoles, bracts and 
accompanying petioles of female plants. Although sufficiently rare on male plants to be 
thought totally absent by some researchers (Hammond and Mahlberg, 1977), their 
existence has been confirmed by others (Dayanandan and Kaufman, 1976). During 
studies for this study, capitate stalked trichomes were only found on males of a few 
varieties and these were restricted to the filaments bearing the anthers. Capitate 
stalked trichomes were the most complex. As shown in Figure 3.6, they developed a 
resin head, similar to that of the sessile type, but in mature specimens this was 
surmounted on top of a multicellular stalk. As experienced by previous workers 
(Mahlberg et al., 1984) it was not always easy to distinguish between sessile and 
immature glandular stalked trichomes, where the stalk was yet to form. Observations 
confirmed the findings of others (Ledbetter et al., 1975) that resin heads on capitate 
stalked trichomes are typically 70-100 |im in diameter, compared to sessile types which 
were more typically 40-50 |im. The stalk consisted of two distinct cell types. 
Hypodermal cells, at the core of the stalk, formed an active channel through which 
nutrients could be transported to the glandular head from the phloem. This was 
surrounded by a single layer of outer epidermal cells, which was a continuous 
extension of that covering the bract or bracteole surface. 



61 



Chapter 3. Cannabis trichome form, function, and distribution 




Figure 3.6. A capitate stalked trichome (centre) between two cystolythic trichomes. The 
specimen is temporarily dry-mounted and illuminated from below. The secretory cells are out- 
of-focus due to the optical distortion within the glandular head (Potter, D.J.). 

Nineteenth-century studies, using the light microscope, identified a disk of secretory 
cells at the base of the resin head (Briosi and Tognini, 1894). More up-to-date electron 
microscope studies have been performed which examined the morphology of this 
feature (Mahlberg et al., 1984; Mahlberg and Kim, 1991; Kim and Mahlberg, 1991, 
2003). The secretory cell disk was readily found during light microscope studies for 
this thesis, albeit initially in the form of a poorly defined mass of translucent material at 
the base of the glandular head. Viewing techniques had to be adjusted to identify the 
individual secretory cells within the disk. 

As with the sessile type, the resin head on capitate stalked trichomes would swell 
during the early stages of development, as the secretory cells became active. The 
contents of the resin head were clear during the earlier stages of development, but 
would become opaque-white in older specimens. In most cases this would not occur 
until trichomes were at least four weeks old, but the rate of loss of transparency was a 
genotypically-dependent characteristic. In CBG-dominant clones (eg M280, Figure 
3.13) glandular heads would become a dense opaque-white when just a few days old. 
This was an unusual example of where the microscope could be used to help identify a 
cannabis plant's chemotype. Further ageing of most genotypes would sometimes result 
in the resin heads turning brown. This colouration was often seen to commence within 
the disk of secretory cells, and was possibly due to necrosis of these now inactive 
tissues. This browning would continue after plants had been harvested and dried. 

Viewing cellular structures within the trichome and surrounding tissues was frequently 
hindered by their refractive properties. This was especially the case when viewing 



62 



Chapter 3. Cannabis trichome form, function, and distribution 



unmounted specimens. The interior of the glandular resin head was particularly difficult. 
Although the tissue was crystal-clear during the early stages of development, as 
observed with sessile trichomes the glandular head acted as a powerful convex lens. 
When illuminated from below the transmitted light was refracted by the glandular head 
contents. This could lead to a distorted virtual image being formed, which appeared to 
be located outside of the structure, as in Figure 3.7. 



Figure 3.7. Two dry-mounted capitate stalked trichomes viewed in transmitted light. Most of 
the features are out-of-focus. In the right-hand trichome, a crisp view of cells within the 
secretory cell disk appears as an in-focus image. However these appear to be located outside of 
the trichome structure, due to the refractive properties of the resin head (Potter, D. J.). 

Mounting the specimen in an appropriate fluid reduced the aberration, as this 
decreased the difference in refractive index between the specimen's contents and that 
of the surrounding medium, the refractive index (Rl) of air being 1 .0. Initial tests found 
water to be a poor mounting medium. Air bubbles frequently became trapped between 
the hydrophobic waxy plant surfaces and the water. Possibly due to its refractive index 
(1.33), image clarity was relatively poor when samples were mounted in water. 
Glycerol (1.47), hempseed oil (1.47) and conventional immersion oil (1.51) have similar 
refractive indices to those of the main terpenes within the resin head (myrcene 1 .48, 
trans-caryophylene, 1.50) and, possibly because of this, these were found to be more 
suitable mounting media. A 70% v/v aqueous solution of glycerol was less viscous and 
sometimes easier to use. This also offered a slightly lower Rl of 1 .44. 

With appropriate lighting, the disk of secretory cells was readily observed at the base of 
the glandular head, as shown in Figures 3.8a. When dry mounted, the secretory cells 
were poorly defined and appeared dark and translucent. When mounted in glycerol, 
more details of the secretory cell disk could be observed. Viewed in profile, the disk 




Secretory cavity 



Secretory cells 



Resin head 
base 



63 



Chapter 3. Cannabis trichome form, function, and distribution 



appeared as an undulating semi-opaque conglomeration of cells (Figure 3.8b). An 
illustration of a trichome viewed from the same angle, as drawn by Briosi and Tognini 
(1894), is included for comparison (Figure 3.8c). In all three Figures 3.8a-c, the stalk is 
seen to include an inner channel of hypodermal cells surrounded by an outer layer of 
epidermal cells. At the uppermost extremity of the hypodermal channel, Figures 3.8b 
and c show a more opaque 'basal cell'. The Briosi and Tognini illustration also shows 
this basal cell to be surmounted by two of the four stipe cells, the distal surface of 
which acts as a base for the secretory cells. The stipe cells cannot be seen clearly in 
this photomicrograph (Figure 3.8b). 




(a) (b) (c) 

Figure 3.8. (a) A temporarily dry-mounted capitate stalked trichome viewed in transmitted light. 
An irregular arrangement of poorly -defined secretory cells is visible at the base of the glandular 
head (Potter, D.J.); (b) A capitate stalked trichome, temporarily mounted in glycerol and 
viewed in transmitted light (Potter, D.J.), and (c) an illustration of a capitate stalked trichome 
on Cannabis sativa by Briosi and Tognini (1894). 

While observing capitate stalked trichomes mounted in oil, the secretory cells were 
perceived to be completely clear structures. These were located above a layer of more 
opaque tissue (Figures 3.9a and b). Transmission electron microscope (TEM) studies 
have shown the distal part of the secretory cells to contain extensive areas of hyaline 
tissue (Kim and Mahlberg, 2003), which may correspond with the clear secretory cell 
tissue viewed here. Related TEM studies also identified increased levels of thickening 
between the secretory and stipe cells (Mahlberg and Kim, 1991), and similar thickening 



64 



Chapter 3. Cannabis trichome form, function, and distribution 



may explain the presence of the apparently-opaque tissue seen at the base of the resin 
head in these studies. 



100pm 




(a) 



(b) 



Secretory space 
Secretory cells 

Thickened 
glandular head 
basal tissue 

Stalk with outer 
layer of epidermal 
cells and inner 
channel of 
hypodermal cells 



Figure 3.9. (a and b). Similar sized capitate stalked trichomes temporarily mounted under hemp 
oil. The samples are viewed in transmitted light. Possibly because of a similarity in the 
refractive index of the oil and the secretory cell contents, these cells appear clear. The outer 
membrane at the base of the glandular head appears dark and opaque (Potter, D.J.). 



TEM studies of the resin head of capitate stalked trichome resin heads have also 
indicated that the secretion produced by the secretory cells enters the secretory cavity 
in the form of so-called vesicles (Mahlberg et al., 1984). These vesicles are separated 
by a network of fibrils, which have a wall thickness approximately half that of the 
secretory cell membrane. Studies of untreated specimens for this thesis, using the STE 
light microscope only rarely gained a clear view of the vesicles. Indeed it was initially 
postulated that the clusters of clear organelles within the secretory head in Figure 3.9a 
and b were not secretory cells, but secretory vesicles. These would have been 
produced from secretory cells, which were initially perceived to be the dark tissues at 
the base of the secretory head. Although this interpretation of the observation is still 
possible, the size of the clear structures appears too large for them to be vesicles, 
which electron microscope studies have shown to be a few microns in diameter 
(Mahlberg et al., 1984). While viewing these secretory cells from directly above the 
trichome, using 70% v/v glycerol as a mounting medium and with light being 
transmitted from below, more credible identification of secretory vesicles was 



65 



Chapter 3. Cannabis trichome form, function, and distribution 



sometimes possible (Figure 3.10). Illuminated in this manner, the secretory cells and 
the secretory vesicles appeared transparent. To maximise the sharpness if the image 
within the optically-distorting secretory head, the microscope was initially focused on 
the subject and the condenser lens then adjusted to gain maximum clarity. 
Appropriately focused, the reported tally of sixteen radially-arranged secretory cells 
could sometimes be counted. As shown in Figure 3.10, when viewed in profile, the 
disk of secretory cells appeared more opaque. 



Figure 3.10. A contrasting pair of resin heads on capitate stalked glandular trichomes, naturally 
orientated to allow sideways-on (left) and overhead views (right). The specimen is temporarily 
mounted in a 70% v/v aqueous solution of glycerol and illuminated from below (Potter, D..J.).. 

Mahlberg etal. (1984) stated that secretory vesicles form above the secretory cells and 
migrate through the secretory head, depositing some of their contents on the secretory 
cavity's outer membrane, thereby maintaining its strength as it inflates. In their studies 
the outer cuticle of the secretory head was seen to increase eight-fold in thickness as 
the trichome developed. These workers used THC monoclonal antibodies to detect the 
presence of THCA within the secretory head. THCA was only detected when attached 
to the vesicle wall and none was discovered within the vesicle interior. If THCA was 
indeed restricted to the vesicle surface, it would not be possible to achieve the THCA 
concentrations of over 40% w/w found in some cannabis resin samples (Potter et at., 
2008; Hardwick and King, 2008). 

The metabolic activity within the secretory cells disk has been well studied by many 
researchers and is still the cause of some debate. The biosynthesis of monoterpenes 




Secretory cell disk 
viewed in profile 



Directly overhead 
view of secretory 
cells, as seen 
through the 
secretory head 



Secretory vesicles 



66 



Chapter 3. Cannabis trichome form, function, and distribution 



and sesquiterpenes, within the membranes of secretory cells, has been identified as 
occurring in plastids and endoplasmic reticulum respectively (Croteau et al., 1984). 
Mahlberg et al. (1984) suggested that these plastids were also involved in cannabinoid 
biosynthesis. Recent research shows that CBGA, the biosynthetic precursor of THCA 
and other cannabinoids, and THCA synthase enzyme are found within the secretory 
cavity (Sirikantharamas et al., 2005). This suggests that the secretory cavity is not just 
the site for the accumulation of cannabinoids, but also the site of THCA biosynthesis. 
The previous theory that THCA is biosynthesised in plastids within the secretory cells is 
further weakened by the observation that THCA and CBGA are cytotoxic 
(Sirikantharamas et al., 2005). By sequestering the cannabinoids in the secretory cavity 
the secretory cells would be protected from damage. The enzymes for cannabinoid 
synthesis within the secretory cavity would require water and molecular oxygen to 
function. As the hydrophobic terpenes accumulate within the vesicles, the oxygen and 
water would most likely be transported via the vesicle fibrillar wall. However, alternative 
studies suggested that CBGA indeed may be synthesised in the secretory cells, but 
further investigations are needed (Taura etai, 2007). 

The secretory cells were confirmed as being metabolically highly-active sites by the 
use of the vital stain tetrazolium red (Figure 3.1 1a). The cells were the first to take up 
the stain and develop a red colour. A 1% solution stained most trichomes within one 
hour, especially in the case in less-mature trichomes where the glandular head 
contents were still clear. When the glandular head's outer membrane was removed by 
abrasion, to expose the disk of secretory cells, the secretory tissue stained red within a 
few minutes (Figure 3.11b). The glandular cells within intact secretory resin heads of 
more mature trichomes were often unstained, suggesting that metabolic activity had 
ceased. However, detailed study of these cells in more-mature trichomes was 
obscured by the age-related increasing opacity of the glandular head (Figure 3.1 1c). As 
this figure also shows, prolonged exposure to tetrazolium red resulted in staining of the 
trichome stalk, through which metabolites would be transported. Within the stalk's 
hypodermis, the phloem transport of sugars to the secretory cells would be via sieve 
tubes. Although phloem transport requires metabolic energy phloem transport is 
passive, but energy is needed to maintain the living condition of these sieve tubes 
(Clifford, 2004). Respiration would also continue in the surrounding epidermal cells, 
resulting in some reduction of the stain. 



67 



Chapter 3. Cannabis trichome form, function, and distribution 



100u.m 





(a) (b) (c) 

Figure 3.11. (a) Secretory cells stained red, within the glandular head, after thirty minutes in 1% 
tetrazolium red; (b) A capitate stalked trichome with glandular head removed by slight abrasion. 
After thirty minutes in 1% tetrazolium the disk of secretory cells is stained bright red; (c) A 
mature capitate stalked trichome between two non-glandular cystolythic trichomes, viewed after 
twelve hours in 0.1% tetrazolium solution (Potter, D.J.). 

As the trichomes aged, it was common for the resin head to become detached from the 
stalk. This would usually arise as a result of the entire head parting with the stalk (as 
commenced in Figure 3.12a). In this photomicrograph the epidermal cells have pulled 
away from the resin head, the hypodermal cells maintaining contact via a narrow 
channel of stipe cells, which are still connected to the disk of secretory cells within the 
resin head. In Figure 3.12b the resin head has completely detached and been 
removed. The stipe cells are just seen protruding from the top of the stalk. Less 
frequently, the resin head would detach as a result of a fissure apparently forming 
above the secretory cell disk (Figure 3.1 3). 



68 



Chapter 3. Cannabis trichome form, function, and distribution 




(a) (b) 

Figure 3.12. (a) A capitate stalked trichome temporarily dry-mounted and viewed in transmitted 
and incident light. The glandular head has become partly detached from the stalk to expose the 
stipe cells, which connect the disk of secretory cells to the hypodermal cells within the stalk; 
(b) The stalk of a capitate stalked trichome after detachment of the resin head. The specimen 
was dry-mounted and illuminated with incident light. The stipe cells can just be seen protruding 
from the top of the stalk (Potter, D.J.). 




Figure 3.13. Separation of the glandular head during (left) and after (right) the appearance of a 
fissure above the secretory cells. The example shown was observed on (M280). The specimen 
was viewed in incident light (Potter, D.J.). 

Figures 3.14a and 3.14b show an alternative view of a capitate stalked trichome before 
and after detachment of the resin head. Many of the cells in this clone are naturally 
pigmented red, some of which are on view within the secretory head (Figure 3.14a). 
During secretory head detachment, the base of the secretory cell disk has remained 



69 



Chapter 3. Cannabis trichome form, function, and distribution 



attached to the stalk, but the secretory cells have been detached along with remainder 
of the secretory head (Figure 3.14b). The secretory head tissue remaining attached to 
the stalk would appear to be formed of thickened resin-head cuticle. In an earlier 
publication it was stated that the red structures within the secretory head were the 
secretory cells (Potter, 2004). With the improved knowledge of trichome morphology 
since gained, it would now appear that these flavonoid-pigmented structures were not 
secretory cells but the vessels carrying carbohydrate and water to the secretory cells. 
These appear embedded in and surrounded by a dense opaque material. A substantial 
proportion of the weight of the resin head clearly has to be attributed to this rigid basal 
material. It is quite likely that much of this material is made of cutin, suberin or both. 
The lateral walls of glandular trichomes in many species become thickened with this 
material, forming a so-called impermeable casparian strip. This prevents apoplastic 
flow of secretory products down the trichome stalk (Werker, 2000). 




(a) (b) 

Figure 3.14. (a) An intact glandular stalked trichome of a naturally pigmented clone Ml 86, with 
coloured cells visible within the resin head. The sample was temporarily mounted in hemp oil; 
(b) A direct overhead-view of the stalk of a capitate stalked trichome on clone Ml 86 after resin 
head detachment. The resin head has become detached leaving thr base of the secretory head 
attached to the stalk. The sample was temporarily mounted in oil (Potter, D.J.). 

Steady views of the outer surface of the base of more-mature secretory heads were 
made possible by trapping the structures on the surface of adhesive tape. (Attempting 
to remove resin heads from very immature capitate stalked trichomes usually ruptured 
the thinner outer membrane, causing spillage of the contents.) The scars of 
attachment to the four stipe cells were readily observed (Figure 3.15). This is 
surrounded by the apparently-encrusted material of the secretory head outer wall. It 
would appear from these studies that there are three methods of secretory head 



70 



Chapter 3. Cannabis trichome form, function, and distribution 



detachment. In the vast majority of cases it would result from a split occurring in the 
basal or stipe cells. Less commonly however, some of the secretory head could remain 
attached to the stalk, and this may or may not include the secretory cells. Whether or 
not the secretory cells are included in the collected material, this would not affect the 
balance of the main secondary metabolites, as research suggests that the secretory 
cells contain no cannabinoids (Mahlberg et al., 2004; Sirikantaramas, 2005; Taura et 
al., 2007). 



Figure 3.15. A detached resin head (approximately 100 um diameter) from a capitate stalked 
trichome, viewed from below to gain a clear view of the scar where the stipe cells were 
originally attached. The head had been trapped on the surface of clear adhesive tape 
(Potter, D.J.). 

On the female plant's calyx, bracteoles, bracts and associated petioles capitate stalked 
trichomes would commonly form a dense pubescence (Figure 3.16a), which would act 
as a physical barrier to small phytophagous insects. By trapping a layer of air close to 
the surface, it would also provide some protection against desiccating cold winds 
(Mahlberg et al., 1984). By reflecting infra-red light a dense trichome pubescence has 
cooling properties and, being equally effective across the complete light spectrum, it 
also reflects ultra-violet (Roberecht and Caldwell, 1980). Phenolic resins like the 
cannabinoids have also been shown to offer UV protection (Rhodes, 1977). This is 
especially welcome in floral structures housing gametophytic tissues, which are 
susceptible to damage by UV-B radiation (Caldwell et al., 1983). Although glandular 
trichomes never exhibited a complete cover on bracts and bracteole tissue, UV 
protection could never be complete, but any degree of protection would improve 
survival chances. 




Scar of attachment 
to stipe cells (four in 
number). 



Ring of heavily 
thickened cell wall 



tissue beneath 
secretory cells. 



71 



Chapter 3. Cannabis trichome form, function, and distribution 



Struggling insects were frequently found trapped to the resin heads of capitate stalked 
trichomes, thereby inhibited from further feeding and reproduction (Figure 3.16b). This 
defensive role of trichomes is observed in many other plant species, e.g. Alfalfa weevil 
Empoasca fabae on lucerne Medicago sativa (Shade et al., 1975) and other insect 
species on Pelargonium spp. (Harman etal.,. 1991).The most common victim observed 
struggling on Cannabis was the cotton melon aphid Aphis gossypii. When attacked by 
predators, Aphis gossypii emits an alarm pheromone to warn other others of danger 
(Byers, 2005). It is possible that a trichome-ensnared aphid responds similarly. One of 
the most common pests of cannabis - the tobacco thrip Thrips tobaci - would also 
become trapped. It too is capable of emitting an alarm pheromone (Anathakrishnan 
1993). If this theory is correct, the loss of a few trichomes to insects could discourage 
a more extensive attack. Restricted allocation of capitate stalked trichomes to floral 
tissue is widespread throughout the Plant Kingdom, where plants optimise investment 
in defence by allocating secondary metabolites to tissues in direct proportion to their 
value (Herms and Matson, 1992). It was notable that sessile trichomes played no part 
in insect entrapment, suggesting that these had a different function. The cannabinoids 
CBGA and THCA have been shown to cause apoptosis in insect cells, and it has been 
suggested that this is an important defensive role for cannabinoids in capitate stalked 
and sessile trichomes (Sirikantaramas etal., 2005). 




(a) (b) 

Figure 3.16 (a) A dense pubescence of glandular stalked trichomes on a bract within a cannabis 
female inflorescence. The specimen was illuminated from behind and photographed with a 
tripod-mounted camera incorporating a macro lens. The orange/brown structures are senesced 
stigmas; (b) two young cotton-melon aphids Aphis gossypii. All six legs on each specimen are 
irreversibly adhered to the resin heads of capitate stalked trichomes (Potter, D.J.). 



72 



Chapter 3. Cannabis trichome form, function, and distribution 



3.5.1.6 Bulbous Trichomes 

With a diameter of approximately 10-20 |im, these were the smallest of the glandular 
trichomes (Figure 3.17a). First seen on the stem and the lower leaves, these were 
widespread across the entire surface of the aerial part of the plant. Connected to the 
epidermis by two cells (the top one much larger than the lower) these would produce a 
simple spherical glandular head (Figure 3.17b) or a rarer complex multi- 
compartmented glandular head (Figure 3.17c). Their function is not known. It was 
notable that the CBG chemovar, shown in Figure 3.13, produced opaque white capitate 
stalked and sessile trichome resin heads. This opacity is attributed to the CBGA. The 
bulbous trichomes on this chemovar were clear or brown, as in Figure 3.16b. This 
suggests that, in this genotype at least, the bulbous trichomes do not contain significant 
concentrations of the cannabinoid. 




(a) (b) (c) 

Figure 3.17 (a) A small bulbous trichome (left) alongside a fully developed glandular stalked 
trichome. The contrast in resin head diameter (10 um v 100 um) is clear; (b) a simple bulbous 
trichome and (c) a complex bulbous trichome. These are 10-15 um in diameter. These samples 
were temporarily dry-mounted and viewed in mixed transmitted and incident light 
(Potter, D.J.). 

3.5. 1. 7 Effect of age and storage on glandular trichome colour 

Glandular trichome resin heads were observed to turn brown as the plant aged, as 
previously reported by Turner et al. (1977) and Mahlberg et al. (1984). Bulbous 
trichomes were the first to discolour, as seen in Figures 3.16b and c. In the glasshouse 
this browning would rarely be seen on capitate stalked trichomes that were less than 
five weeks old. The colouration continued during storage at ambient temperatures. 
The clear colouration of freshly harvested floral material is contrasted with the brown 
colour of a three-year old sample in Figures 3.18a and 3.18b. The width of the 



73 



Chapter 3. Cannabis trichome form, function, and distribution 



glandular stalk in the dried sample has narrowed during drying due to the collapse of 
the epidermal cells. This has also resulted on the stalks losing their turgid upright 
appearance. 




(a) (b) 

Figure 3.18. (a) Clear glandular stalked trichomes on freshly harvested young cannabis floral 
tissue; (b) Brown trichomes on three-year old stored cannabis (Potter, D.J.). 



3.5.2. Effect of glandular trichome array on the secondary metabolite 
content of plant tissues 

The densities of capitate stalked and sessile trichomes counted on the proximal and 
distal sections of bracts are shown in Figure 3.19. 




Proximal Stalked Proximal Sessile Distal Stalked 

Trichome Type and Distribution 



Distal Sessile 



Figure 3.19 The mean density of capitate stalked and sessile trichomes (± 1SE) (upper and 
lower surfaces combined), on each of three high-THC cultivars M3, M6 and M7. On each clone 
twenty randomly selected fields were counted on both the upper and lower surface in the 
proximal and distal areas. 



74 



Chapter 3. Cannabis trichome form, function, and distribution 



This confirmed that the bract dissection method had been successful in separating the 
samples into proximal sections, where capitate stalked trichomes were abundant, and 
distal sections where they were absent. Despite the polarisation of capitate stalked 
trichome populations, the density of sessile trichomes was found to be similar in 
proximal and distal areas. It was also noted that glandular stalked trichomes were in 
the greatest numbers on the upper surface of the bract in this variety. Studies with 
other varieties (not reported here) showed trichome density to be greatest on the 
underside of the bract. 

In all three clones analyses of variance showed that distal tissue had a significantly 
higher proportion (p < 0.001) of CBC within the cannabinoid profile. Figure 3.20 
showed that the capitate stalked trichomes in the proximal area outnumbered sessile 
trichomes by a factor of approximately five. Individual capitate stalked trichome resin 
heads have a volume approximately eight times greater than that of a sessile trichome. 
Combining these factors, the potential difference in volume of the capitate stalked and 
sessile populations is therefore a factor of forty. The findings of previous workers 
suggest that the difference in quantity of cannabinoid stored could be even greater, as 
capitate stalked trichome contents were also found to have a higher cannabinoid 
concentration (Turner et at., 1977). This suggests that, on tissues that have abundant 
capitate stalked trichomes, the sessile trichomes population would have minimal 
influence on the overall cannabinoid potency and profile of that tissue. 



25 

o 

CQ 

O 20 
+ 

o 

15 



O 

o 



10 



M3 



M6 



M7 



h 



Distal 
Proximal 



Mean 



Clone 



Figure 3.20. The proportion of CBC expressed as % of THC+CBC in the cannabinoid profile of 
proximal and distal tissue of bracts from each of three high-THC clones. (Error bars on clones 
represent ± 1SD, and on the mean ± SE). In all three clones the difference was highly significant 
(ANOVA, *** denotes p < 0.001). 



75 



Chapter 3. Cannabis trichome form, function, and distribution 



Producers of illicit sinsemilla cannabis commonly recognise that distal bract tissue has 
a much lower cannabinoid content. It is common practice to remove the distal part of 
the less resinous bracts. The material that remains is consequently reduced in weight, 
but its overall potency is increased. Illicit producers commonly refer to this process as 
'manicuring' (Potter, 2004). The practice has also been followed by producers of 
pharmaceutical grade medicinal cannabis in the Netherlands (Institute of Medical 
Marijuana, 2008). In pharmacognosy, the removal of less desirable material from a 
pharmaceutical feedstock is sometimes called 'garbling' (Tyler etal., 1988). This study 
suggests that, where bracts only exhibit glandular stalked trichomes on the proximal 
tissue, removal of the distal tissue will indeed increase the potency of the material that 
remains. The cannabinoid profile of the remaining material will be minimally affected, 
because of the relatively small influence of the associated sessile trichome population. 

3.5.3 Effect of capitate stalked trichome density and colour on 
cannabinoid content and profile. 



The mean THC content was calculated for all sinsemilla samples attracting the same 
score for trichome density. The vast majority of samples attracted scores on the first 
five points of this 1-9 scale (76% being awarded a score of 2 or 3) and meaningful 
standard error calculations were only possible on values in this range (Figure 3.21). 





18 




16 




14 




12 


X 


10 


1- 


8 




6 




4 




2 









1 

14 



2 


3 


4 


5 


6 


7 


8 


64 


126 


32 


6 


3 


2 






1-9 Score 
n 



Figure 3.21 The correlation between capitate stalked trichome density (visually assessed 1-9 
scale, 1 = high, 9 = low) and the overall THC content of the sample. Values shown are the 
mean % w/w THC content (± SE only where n > 5) of all samples for each density score. 
Regression line is shown in red. The regression model is:- % THC = 18.051 - 1.629 * Density 
Score. (p< 0.001, R 2 = 0.17). 



76 



Chapter 3. Cannabis trichome form, function, and distribution 



The results show that there is clearly a significant tendency for plant samples with 
greater capitate stalked trichome densities to have a higher cannabinoid content. In 
plants with a less dense pubescence, high cannabinoid contents are not simply 
maintained by more secondary metabolite being sequestered in each trichome. A high 
density of glandular trichomes is therefore a very useful visual parameter upon which a 
judgement can be made of the cannabinoid content of dry plant material. However, the 
low R 2 value shows that this is only an approximate guide when assessing samples of 
variable genotype and provenance. 

The relationship between trichome colour and cannabinoid content is shown in Figure 
3.22. The data were more evenly spread than those from the corresponding trichome 
density study. The slope of the regression line shows that there is a weak but 
significant tendency for darker coloured trichomes to be associated with low-potency 
cannabis samples. Turner et al. (1977), comparing the THC content of colourless and 
brown capitate stalked trichomes of a single clone, reported that brown trichomes 
contained less THC per trichome. However, there were no accompanying statistical 
analyses to clarify the significance of the reported finding. 




Figure 3.22. The correlation between capitate stalked trichome colour (visually assessed using a 
1-9 scale, 1 = clear, 9 = darkest brown) and the overall THC content of the sample. Values 
shown are the mean % w/w THC content (+SE) of all samples for each colour score. 
Regression line is shown in red. The model for this is: - Percent THC = 15.990 - 0.587 * 
Trichome Colour Score (p < 0.001, R 2 = 0.053). 



77 



Chapter 3. Cannabis trichome form, function, and distribution 



The pigment causing this brown colouration is not known. Commencement of browning 
was commonly observed in the region around the secretory cells and could be due to 
oxygen entering the resin head via the scar tissue (Figure 3.13) when it becomes 
detached from the epidermal cells on the trichome stalk, as shown in Figure 3.12a. 
Alternatively it could be caused by the biproducts of catabolism of the secretory cells 
contents. 

The mean average CBN content of the samples awarded each score is shown in 
Figure 3.23. For those samples where CBN contents were very low and below the 
minimum detectable level of 0.10% a nominal value of 0.05% was used. 




Figure 3.23. The mean CBN content (± SD) of populations of sinsemilla samples awarded each 
of the 1-9 ratings for trichome colour 1 = clear, 9 = darkest brown. All samples were seized by 
police in 2004/2005. 



There was a trend for samples with darker trichomes to have a higher CBN content but 
the data was extremely variable, as shown by the large standard deviations. The 
degree of THC catabolism was assessed by expressing the CBN as a proportion of the 
CBN+THC total for each sample (i.e. CBN/(THC+CBN). The correlation between 
trichome colour and CBN/(THC+CBN) is shown as a scatter-graph in Figure 3.24. This 
shows that very little CBN existed in clear or white trichomes. A high level of CBN 
formation was restricted to plants with brown glandular trichomes, but dark coloured 
trichomes could still be devoid of this catabolite. Six values were identified as clear 



78 



Chapter 3. Cannabis trichome form, function, and distribution 



outliers, according to the ISO recommended Grubb's Test (Miller and Miller, 2005) and 
excluded. 




Figure 3.24. The variability in degree of THC catabolism to CBN as related to their colour. Of 
249 original samples 6 were rejected as outliers and not included here. Trichome colour was 
assessed visually and scored on a 1-9 scale where 1 represents totally clear and successively 
higher scores denote an increasing opacity and darkening in colour. 



The comparison between trichome colour and CBN content showed that the lower 
average potency in darker colour trichomes could be almost entirely due the THC 
having catabolised in these samples. There was a greater likelihood of finding CBN in 
brown coloured trichomes but its presence was not guaranteed. This shows that if a 
pharmaceutical company demands cannabis material containing minimal levels is 
CBN, a brown colouration does not immediately imply rejection of the sample. 

It appears that the conditions required for the oxidative catabolism of THC to CBN are 
not the same as those causing the brown colouration. Experienced observation of 
many samples has shown that this pigmentation is not reversible and the colour 
stabilises. Indeed, this colour was seen to have been maintained in trichomes 
photographed while contributing to studies on an archaeological cannabis sample 
discovered in an ancient Chinese tomb. Carbon dating estimates the age of the 
sample in Figure 3.25 as 2700 years old. It is accepted that additional catabolic 
reactions would have occurred over time and possibly had an influence on the colour, 



79 



Chapter 3. Cannabis trichome form, function, and distribution 



but catabolism appeared to have stabilised and cannabinoids were still present in these 
trichomes (Russo etal., 2008). 




Figure 3.25. Aged brown sessile glandular trichomes on 2700 year old cannabis. The sample is 
illuminated with incident light and photographed digitally. The pubescence of unicellular non- 
glandular trichomes is also clearly visible (Potter, D.J.). 

3.5.4 Effect of photosynthetic ability, or lack of ability, on cannabinoid 
biosynthesis in sessile trichomes on variegated leaf tissue. 

The potency of the leaf tissue samples in these tests is shown in Table 3.4. On a dry 
weight basis, yellow leaf material was clearly more potent than green. This would have 
been influenced by the fact that green tissue weighed 33% more per unit area, 
probably due to the production and storage of starch in these tissues following 
photosynthesis. By assessing potency in terms of cannabinoid per unit area, the effect 
of starch accumulation would be removed. Assessed this way, the yellow material was 
still significantly more potent (p < 0.05), although the difference was much smaller. 
Microscope observations suggested that this difference was not due to differences in 
densities of sessile trichomes on yellow and green tissue, but an accurate count was 
not possible because of the density of non-glandular trichomes obscuring the view. 
The earlier work by Crombie (1977) had shown that cannabinoid biosynthesis still 
continued in 'albino' tissue. This study went further and suggested that cannabinoid 
biosynthesis was totally unrelated to the immediate tissue's ability to photosynthesise. 
However, the rate of biosynthesis in these tissues would be affected by the overall 
photosynthetic-ability of the plant. 



80 



Chapter 3. Cannabis trichome form, function, and distribution 



The biosynthesis of terpenoids has a higher energy requirement than most other 
primary and secondary metabolites, because of the extensive level of chemical 
reduction within these compounds (Gershenzon, 1994). The energy for this 
biosynthesis would be derived from light energy, captured within the chloroplast. 
During periods of light exposure, some chloroplasts would be directly illuminated and 
others would be in varying degrees of shade. This study showed that plant tissues 
devoid of chlorophyll, and thereby unable to photosynthesise, could support the 
biosynthesis of cannabinoids within their own glandular trichomes. The carbon source 
required for cannabinoid biosynthesis would have been produced elsewhere within the 
plant and then translocated to these trichomes. This demonstrates that in a normal 
growing environment, where some parts of the plant are in full sun and others in 
varying degrees of shade, all aerial parts the plant will be able to synthesise 
cannabinoids. The resultant increase in trichome content uniformity is fortunate for the 
grower of cannabis for the pharmaceutical industry. 





Green Tissue 


Yellow Tissue 


ANOVA 
p value 


Potency by Weight Test 1 . THC % w/w 
dry (n = 3 bulked samples) 


1.51 (±0.26) 


2.30 (± 0.26) 


0.008 


Potency by Weight Test 2. THC % w/w 
dry (n = 2 bulked samples) 


1.18 (±0.08) 


1.92 (±0.08) 


0.005 


Potency by Area Test 2. THC g m" 2 
(n = 2 bulked samples) 


0.50 (± 0.03) 


0.61 (±0.01) 


0.038 



Table 3.4. The potency (THC content) of yellow and green leaf tissue of the variegated cultivar 
G60-M55 assessed in each of two tests (± SD). The potency in the second test is also shown as 
a weight of THC per unit area. 



Mahlberg etal. (1983) compared the cannabinoid content of capitate stalked trichomes 
on the upper and lower surfaces of cannabis bracts and found those on the upper 
surface to be more potent. This was attributed to the upper surface receiving more 
light. This study with variegated cannabis weakens that argument, showing that the 
ability of leaf tissue to photosynthesise has minimal effect on its ability of the local 
glandular trichomes to synthesise cannabinoids. 



81 



Chapter 3. Cannabis trichome form, function, and distribution 



3.6 CONCLUSIONS 

While performing a detailed study of cannabis trichomes, Dayanandan and Kaufman 
(1976) stated that the study of these organelles was essential to understand the 
biogenesis, distribution and function of the different cannabinoids. The research 
reported in this chapter clearly showed how the distribution of THC and CBC differed. 
The subcellular details of cannabinoid secretion were also shown in greater detail than 
perhaps previously captured with light microscopes. 

Within the pubescence on Cannabis sativa L there are six forms of trichome, of which 
only five are found on the female plant. With appropriate mounting and staining, it was 
shown that what was believed to be a previously unavailable undistorted views of the 
internal structures of these trichomes could be differentiated to a high degree with the 
use of low cost microscopes. The use of these mounting media could facilitate future 
further studies of these structures. 

It was shown that the cannabinoid profile of bract tissue appears to be affected by the 
ratio of so called 'capitate stalked' and 'sessile' trichomes present. This suggests that 
the two types of trichome had differing cannabinoid profiles. By separating tissues with 
differing ratios of capitate stalked and sessile trichomes, it was shown that the grower 
had the capability to produce samples with significantly different cannabinoid profiles. 
The research also showed that insects were regularly ensnared by glandular stalked 
trichomes but never by capitate sessile trichomes. This was a clear indication that 
these two trichome forms differed in function. It was hypothesised that this was 
possibly attributable to a difference in secondary metabolite content. The observed 
contrasting sizes and densities of glandular stalked and sessile trichomes suggested 
that in floral material, exhibiting both trichome types, the capitate stalked trichome 
population had a potentially greater influence on cannabinoid profile than the sessile 
trichome population. 

Vital staining techniques provided supportive evidence that that the secretory cells 
within the trichome head were the area of greatest metabolic activity. Novel studies 
with variegated cannabis also showed that secretory cells within leaf tissue lacking 
chlorophyll suffered no reduction in cannabinoid synthesis ability. In fact a small but 
significantly higher concentration of cannabinoid (weight per unit area) was found in 
tissue lacking chlorophyll. Although the overall ability of the plant to biosynthesise 
cannabinoids was likely related to the total amount of energy available to the plant, this 
study implied that where part of a plant was in shade, this did not affect its ability to 



82 



Chapter 3. Cannabis trichome form, function, and distribution 



biosynthesise cannabinoids ability. Thus partial shading did not increase the variability 
of plant tissue cannabinoid contents. 

As glandular trichomes age it was observed that there is a colour change, the initially 
clear resin head first turning white and then brown. On feedstock exhibiting brown 
trichomes there is an increased likelihood, but not a guarantee, that there will be a 
reduced THC content and a raised content of the THC catabolite CBN. A high density 
of capitate stalked trichomes on the female floral tissue indicates an increased 
probability, but not a guarantee of high cannabinoid content. 

As the capitate stalked form matures, the resin head is increasing likely to detach itself 
from its stalk. When this occurs, the secretory cells are usually retained within the 
resin head, but they are sometimes left attached to the stalk. This mode of 
detachment would be expected to affect the proportion of waxes, proteins and other 
unidentified ingredients within any enriched trichome preparations made. 

Having assessed the form and distribution of the trichomes, Chapter Four looks in 
detail at the chemical content of the capitate types. It also assesses how this alters as 
trichomes age. The knowledge gained would help the grower identify the optimum time 
for harvest and evaluate the possibly of producing feedstocks by removing these 
trichomes from the plant. 



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Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



Chapter 4. The Function and Exploitation of Secondary 
Metabolites from Glandular Trichomes of Cannabis sativa L. 

4.1 INTRODUCTION 

Across the Plant Kingdom, glandular trichomes are responsible for the biosynthesis 
and/or sequestration of a vast range of secondary metabolites. Of these the terpenoids, 
which includes the cannabinoids and cannabis-related terpenes, are the most common 
and structurally diverse compound group, and these are spread throughout many plant 
families (Kelsey et al., 1984). Other carbon, hydrogen and oxygen based categories of 
secondary metabolites are also found, but are restricted to the glandular trichomes of 
smaller ranges of species. Examples include the methylated flavonoids of the creosote 
bush Larrea tridentata (Thompson et al., 1979) and the quinones found in the 

semitropical plant family Hydrophyllaceae (Kelsey , 1984). 

The total number of secondary metabolites found in plants has been estimated at 
approximately thirty thousand. Of these, about 40% contain nitrogen, of which all but a 
thousand belong to the alkaloid group (Acamovic and Brooker, 2005). Of the secondary 
metabolites found in glandular trichomes, the proportion that contains nitrogen is much 
less than 40% (Wagner etal., 2004). Examples include histamine, which is synthesised 
within the stinging glandular trichomes of nettles Urtica spp. Another example of great 
importance to world health is the alkaloid nicotine, which is abundant in trichomes of 
some cultivated varieties of tobacco Nicotiana tabacum. Although sequestered in 
trichomes, this alkaloid is actually translocated there from the roots where it is 
biosynthesised. Trichomes of the velvet bean Mucuna pruriens also contain alkaloids 
and additionally produce serotonin (5-hydroxytryptamine) (Ghosals Singh etal., 1971). 

The previous chapter described the different forms of non-glandular and glandular 
trichomes in Cannabis sativa L and compared their differing functions. It also reported 
studies which supported previous researchers' findings (Turner et al., 1977, Mahlberg 
et al., 1984) that capitate stalked and sessile trichomes differed in their cannabinoid 
profile. It was shown that by segregating plant tissues, according to their trichome 
pubescence characteristics, it was possible to produce feed-stocks with significantly 
different cannabinoid profiles. This chapter looks in greater detail at the secondary 
metabolite content of these trichomes. It was hypothesised that, if the sessile and 
capitate stalked trichomes could be removed from the plant and then separated, it 
might be possible to provide feedstocks with more closely controlled cannabinoid 



84 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



profiles. Separating trichomes that were at different stages of maturation may also 
have benefits. 

The removal of glandular trichomes from living Cannabis plants is common practise, 
being a major part of cannabis resin manufacture. Many centuries-old cultural methods 
are practised, but few are of relevance to the modern pharmaceutical industry. One 
method of resin manufacture includes manual rubbing of the inflorescences, resulting 
in the trichomes adhering to the hands (Cerniak, 1985). British physician W.B. 
O'Shaughnessy, who introduced cannabis to Western medicine from India, reported 
that workers dressed in leather would walk through the crop and then scrape off the 
resin that adhered to their clothing (Samuelsson, 1999). Both techniques rupture the 
trichomes, accelerating the loss of the more volatile components and the oxidation of 
others. The materials also become heavily contaminated with other plant fragments. 
More common methods of trichome removal for resin production are based upon the 
sieving of dried cannabis plants. This practise is utilised in Morocco, which supplies the 
vast majority of cannabis resin entering the UK (UNODC, 2006). Plants are dried and 
sometimes left for many weeks before processing, by which time the trichomes will 
have lost much of their more volatile terpene content. Techniques used to make so- 
called 'modern hashish' (Clarke and Watson, 2007) perhaps offer greater opportunities 
for collecting undamaged glandular trichomes, with more of their secondary 
metabolites intact. In one method, cannabis material is simply agitated in cold water 
and the dislodged capitate stalked trichomes collected using sieves. Jansen and Terris 
(2002) reported that, with a Dutch Government subsidy, this technique had been 
adapted to make hashish for pharmaceutical research purposes. Separation of 
capitate stalked and sessile trichomes was not reported. For the remainder of this 
thesis, the terms hashish and resin are avoided when referring to products made for 
scientific as opposed to recreational purposes. The term enriched trichome preparation 
(ETP) is preferred. 

As well as the cannabinoids, most of the monoterpenes and sesquiterpenes found in 
Cannabis are also located in the glandular trichomes (Malingre, et al., 1975; Turner et 
al., 1980). These are generally present in much smaller quantities than the 
cannabinoids, and this hinders their detailed study. Studies of the terpene content of 
cannabis trichomes are few and have involved the steam distillation of intact cannabis 
tissue as the source material (Mediavilla and Steinemann, 1997). The bulk collection of 
intact cannabis trichomes would clearly facilitate more detailed terpenoid analysis by 
providing a richer source of material for analysis. To enable this to happen, research 
was needed to assess the feasibility of removing and then segregating large quantities 



85 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



of intact glandular trichomes from cannabis. A number of techniques have been 
employed to remove intact glandular trichomes from other species. These have been 
reviewed by Wagner et al. (2004). Techniques included removal of individual trichomes 
with forceps, shaking in aqueous solution with an abrasive (e.g. sand, fine glass beads 
or powdered dry ice) and the gentle brushing of fresh or frozen tissue. In some cases 
different trichome forms have subsequently been separated by sieving and/or 
centrifugation, e.g. separation of bulbous and peltate trichomes of sweet basil Ocimum 
basilicum (Gang et al., 2001). All these collection methods produce extremely small 
trichome samples. In contrast, removal of glandular trichomes on an industrial scale is 
currently performed in the hop (Humulus lupulus) industry. By agitating plant material 
in extreme cold temperatures, the sessile glandular trichomes (known commercially as 
lupulin) are dislodged and separated from the plant (Rigby, 2000). 

The previous chapter confirmed the findings (Ledbetter and Krikorian, 1975) that the 
four types of glandular trichome found in Cannabis, namely bulbous, capitate sessile, 
antherial sessile and capitate stalked, had diameters of approximately 10 urn, 50 urn, 
80 urn and 100 urn respectively. Based on their comparative spherical dimensions a 
bulbous trichome would have a potential secondary metabolite content approximately 
one hundred and twenty five times less than that of a capitate sessile trichome. 
Previous workers have expressed difficulties in working with bulbous trichomes 
because of their small size, and evidence for the existence of cannabinoids in these 
trichomes is lacking. Regular observations of bulbous trichome populations, while 
studying other trichome forms for this thesis, suggested that the population densities of 
bulbous trichomes were similar to those of sessile trichomes. This supported the 
observation of previous researchers (Turner ef a/.,1978; Mahlberg et al., 1984). This, 
combined with the minute size, suggests that bulbous trichome contribute little to the 
overall secondary metabolite content of cannabis. Bulbous trichomes were not studied 
further for this thesis. 

4.2 AIM AND OBJECTIVES 

A series of studies were performed with the aim of separating, purifying and analysing 
substantial quantities (>1g) of intact glandular trichomes from cannabis. It was 
envisaged that trichome filtrates derived from cannabis tissues of differing chemotype, 
stage of maturity and location on the plant might exhibit different secondary metabolite 
profiles. As progress was made the following sequence of objectives was developed: - 

4.2.1. To collect and separate intact capitate stalked and sessile glandular trichomes 
from fresh floral material. 



86 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



4.2.2. To isolate intact sessile glandular trichomes from vegetative material. 

4.2.3. To collect sessile trichomes from foliage of a high-CBC chemotype as a means 
of isolating the minor cannabinoid CBC 

4.2.4. To examine the ontogenetic changes in terpenoid content of glandular trichomes 
during plant maturation. 

4.3 MATERIALS 
4.3.1 Germplasm 



Clone 


Chemotype 


Variety Name 


Supplier 


G1-M3 
G2-M6 
G5-M13 
G5-M16 
M240 


High-THC 
High-THC 
High-CBD 
High-CBD 
High-CBC 


Guinevere 
Galina 
Grace 
Gill 

Unnamed 


GW Pharmaceuticals Ltd, 

Porton Down Science 
Park, Salisbury, Wiltshire. 



Table 4. 1 Name, source and chemotype of clones used. 



4.3.2 Apparatus 



Kenwood HM 220 Food Mixer 


Dixons Ltd. Internet purchase. 


25 urn, 43 urn, 73 urn and 
220 urn 'Bubblebag' Sieves 


EveryoneDoesIT Unit D1, Phoenix Industrial Estate, 
Rosslyn Crescent, Harrow, HA1 2SP 


Ebb and Flood Irrigation Tank 
2.0 x 0.7 x 0.4 m 


Bridge Greenhouses Ltd., Chalk Lane, 
Sidlesham, Chichester, PO20 7NQ 



Table 4.2 Miscellaneous items and commercial sources. 



4.4 METHODS 

4.4. 1 Separation of sessile and capitate stalked trichomes glandular heads 
from mature fresh floral material. 

In the first of a series of investigations, this test adapted a method of removing 
trichomes from cannabis material described by Jansen and Terris (2002). The high- 
THC clone G1 M3 was used for this study. Approximately 500 g of fresh floral material 
was removed from a batch of fully mature plants. These had been in flower for eight 



87 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



weeks and were at the normal stage for harvesting. Using scissors, the material was 
cut into pieces, each being no greater than approximately five grams. Six randomly 
selected pieces were retained for analysis. The material was then plunged immediately 
into twenty five litres of slurry containing tap water and crushed ice. The mixture was 
gently mixed for ten minutes to allow the plant material to cool and then agitated for ten 
minutes using a Kenwood HM220 domestic food-mixer operating at maximum speed. 
Manual examination confirmed that the glandular trichomes lost their sticky texture at 
low temperatures. A large proportion of the trichome glandular resin heads were 
dislodged from the plant by this process. Being denser than water they sank and 
readily separated from the pulp when poured through a fine sieve (220 urn approximate 
mesh). The resin heads passed through the mesh and the 'spent pulp' was retained. 
The resin heads are then efficiently separated from the bulk of the water by pouring the 
suspension through a 73 urn sieve and then a finer 25 urn sieve. In theory, the resin 
heads from glandular stalked trichomes (reported typical diameter 75-100 urn) should 
have been trapped on the 73 urn sieve, sessile trichomes (typically 50 urn) falling 
through and being caught on the 25 urn mesh. 

The resin heads collected from each sieve were removed and a few milligrams taken 
for microscope study. The remainder was frozen while awaiting chemical analysis. 

4.4.2 Bulk-Production of Pure Sessile Trichome Preparations. 

Five kilograms of leaves were removed from plants of the high-THC clone G1 M3. The 
plants had been grown for three weeks in continuous lighting (75 Wm" 2 PAR) at 25°C, 
and were therefore still in the vegetative phase and devoid of capitate stalked 
trichomes. The collected material was thoroughly mixed and approximately twenty 
grams frozen (-20°C) pending analysis by GC (Appendix 2). The 5 kg of foliage was 
thoroughly mixed in a shallow tank with 200 litres of iced water for thirty minutes using 
a domestic food mixer at maximum speed. The bulk of the treated foliage was then 
manually removed and the liquid remains drained through 220 urn and 25 urn sieves. 
The material collected on the second sieve was examined for purity, using a 
microscope, and a sub-sample (approximately 1 g) taken for cannabinoid assay. The 
remaining material was frozen prior to steam distillation and qualitative chemical 
analysis by GC at Botanix Ltd. A batch of plants (~ 300) of the same clone was also 
grown to full maturity, then harvested and dried for routine drug production. Six random 
subsamples (approximately 5 g) of the stripped leaf and foliar material were thoroughly 
mixed prior to analysis by GC (Appendix 1). 



88 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



4.4.3 Production of a cannabichromene-rich sessile trichome preparation. 

The production of a CBC-rich sessile trichome preparation was attempted, using the 
above trichome collection method. The plant material utilized was stripped foliage of 
the CBC-rich clone M240. These plants had been grown vegetatively for four weeks in 
the same regime. 

4.4.4 Ontogenetic changes in Secondary Metabolite Content of Glandular 
Trichome Contents. 

Two cultivars were selected for this study; high-THC clone G2 M6, and high-CBD clone 
G5 M13. Plants were grown according to the routine methods for the propagation of 
the medicinal cannabis crop. Ten randomly selected plants of each cultivar were 
removed from the crop after three weeks of vegetative growth. Further samples were 
made on each of five harvest dates - six, seven, eight, nine and ten weeks after the 
induction of flowering. The foliage and floral material from each batch was stripped 
from the stem. This was thoroughly mixed and six subsamples (approximately 10 g 
each) were taken. All materials were frozen at -20°C pending analysis. Stems were 
discarded. The six subsamples were subsequently analysed for cannabinoid content by 
GW Pharmaceutical Ltd using GC (Appendix 1). 

The main samples were distilled by Botanix Limited according to their Standard 
Operating Procedure for the Determination of Volatile Hop Oil (Institute of Brewing 
Method). Each was ladled into a one-litre round bottom flask with a B55 neck. A few 
anti-bumping granules were added before connecting each one to a 'British 
Pharmacopoeia Still' using a B55/34 adaptor. The flasks were then heated using a 
heating mantle and the contents distilled for three hours. Employing this method, 
Howard (1970) showed that the extraction of essential oils from hop lupulin (glandular 
trichomes) was reliably complete in three hours and indeed no further distillate was 
seen to be collected after this time. During this period the flow of condensate was 
controlled to cause minimum disturbance to the oil in the trap. At the end of three 
hours the volume of oil was recorded and decanted. These oil fractions were analysed 
for qualitative terpene content by Botanix Ltd. The quantitative content of a small range 
of terpenes within this mixture was determined by GC at GW Pharmaceuticals Ltd 
(Appendix 2). 



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Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



4.5 RESULTS AND DISCUSSION 

4.5.1 Separation of Sessile and Capitate Stalked Trichomes Glandular 
Heads from mature fresh floral material 

Table 4.3 shows the cannabinoid content of single trichome preparations collected from 
the fine and coarser sieves and a bulked sample of plant material before and after 
trichome collection. Three sub-samples of each material were taken for analysis. The 
standard deviations therefore only indicate the uniformity of the preparations and the 
precision of the analytical method. 



Sample Details 


% of each cannabinoid in the cannabinoid 
total ± SD 


% w/w total 
cannabinoid 
± SD 


CBC 


CBG 


THC 


Sessile trichome rich 
filtrate - 25 urn sieve 


1.44 ± 0.02 


1.19 ± 0.04 


97.37 ± 0.05 


37.00 ± 7.26 


Capitate stalked trichome 
rich filtrate - 73 urn sieve 


0.93 ± 0.02 


0.86 ± 0.03 


98.21 ± 0.02 


58.70 ± 4.29 


Starter Material 


1.13 ± 0.14 


1.30 ±0.05 


97.58 ±0.16 


11.50 ± 0.34 


Spent Pulp 


1.07 ± 0.18 


1.21 ± 0.03 


97.72 ± 0.19 


7.30 ±1.53 



Table 4.3. A comparison of the cannabinoid profile and content of fresh cannabis floral 
material (clone Gl M3) and sieved trichome filtrates made there from. One sample of each 
fraction was produced. Analyses show mean analytical results of three subsamples (±SD). Also 
shown is the cannabinoid content of the floral material before trichome extraction and the spent 
pulp after extraction (n=3). 



Microscope analysis confirmed that most of the material caught on the 73 urn sieve 
consisted of the larger resin heads from capitate stalked trichomes. The material 
caught on the 25 urn sieve appeared to consist predominantly of the smaller sessile 
trichome resin heads. However, the resin heads from some immature capitate stalked 
trichomes may have also passed through the 73 urn sieve and been included here. 
The material collected on the finer 25 urn sieve had a much lower cannabinoid content 
(37% w/w) than that removed from the coarser 73 urn sieve (59% w/w). This supports 
previous observations in a previous study that showed the cannabinoid concentration 
in individually collected sessile trichomes to be lower than that of the capitate stalked 
form (Turner etai, 1978, 1979). 



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Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



The sessile trichome rich filtrate on the finer sieve had a notably higher proportion of 
the cannabinoid CBC. The result supports the finding of the previous chapter, 
suggesting that the two trichome forms appear to have different cannabinoid profiles. 
However, in that earlier study, the comparison was made between capitate stalked and 
sessile trichome populations from individual bracts, and the two trichome forms were of 
the same age. In this later study with sieved trichome samples, the average age of the 
sessile trichomes would have been greater than that of the capitate stalked trichomes. 
This is because the sessile form would have been produced throughout the lifetime of 
the plant, whereas the bulk of the capitate stalked trichomes would have been formed 
within the last few weeks of growth. It has been shown that, in most chemotypes, the 
proportion of CBC within the cannabinoid profile of the whole plant drops sharply during 
the life-time of the plant (Shoyama et al., 1975; de Meijer et al., 2009). The drop is 
greatest during the first weeks of growth when no capitate stalked trichomes are 
present, and the decrease in CBC proportion thus cannot be attributed to a changing 
balance of sessile and capitate stalked trichomes. The higher proportion of CBC in the 
filtrate on the finer sieve would have thus likely been due to two factors: - 

i) It contained more sessile trichomes, which have been shown to have a higher 
proportion of CBC to capitate stalked trichomes of the same age, and 

ii) The trichome on this sieve contained a higher proportion developed within the first 
weeks of growth when the proportion of CBC within the whole plant was much higher. 

Table 4.3 also shows that there was a higher proportion of CBG in the cannabinoid 
profile of the material collected on the finer sieve. It is not possible to state if this is due 
to direct differences in the cannabinoid profile of sessile and capitate stalked trichomes, 
or due (at least partly) to the younger average age of the capitate stalked trichome-rich 
material. (Younger material would reasonably be expected to contain a higher 
proportion of trichomes within which less of the GPP and olivetolic acid had been fully 
converted into THC via the intermediate in question - CBG.) In the previous chapter, 
where the cannabinoid profiles of similar aged trichome populations on proximal and 
distal bract tissue were compared, CBG was not present in detectable quantities in all 
samples. A comparison with that test is therefore not possible. Even though the 
reasons were not totally clear, from this test it was important to note that separating 
trichomes with sieves appeared to offer a means of producing phytopharmaceutical 
feedstocks with a favorably altered cannabinoid profiles. 



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Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



4.5.2 Isolation of intact sessile glandular trichomes from vegetative 
material 

This test clearly demonstrated that intact sessile trichome resin heads could be readily 
collected from non-flowering cannabis vegetation. This, and the previous test, 
overcame concerns that the technique might only be efficient on capitate stalked 
trichomes which, by virtue of their greater size and upright shape, would experience 
greater shearing forces from the agitated iced-water. A sub-sample of this preparation 
is shown in Figure 4.1 . 




Figure 4.1. A smeared sample of sessile glandular trichome resin heads prepared from 
vegetative foliage of high-THC clone Gl M3. The freshly captures specimens were collected 
from the surface of a 25 um sieve and are free-floating in water. A few pieces of leaf fragment 
are also present as a minor contaminant. 

It can be seen that there was some minor contamination of this material, with 
fragments of green leaf tissue. The proportion of resin heads within this filtrate was 
higher than that generally produced when collecting resin heads from capitate stalked 
trichomes. The latter would be routinely contaminated with capitate glandular trichome 
stalks (which are not present on foliage) and cystolythic trichomes (which more readily 
pass through the coarser sieve). The cannabinoid content of materials collected from 
both the 25 um and 45 um sieves were analysed. In Table 4.4, this is compared to the 
cannabinoid content of the combined foliar and floral material collected from mature 
plants of the same cultivar. Although THC was still the dominant cannabinoid in the 
sieve contents, it can be seen that the proportion of CBC within the cannabinoid profile 
was greatly increased. The results show the content of trichome preparations collected 
from 45 um and 25 um sieves. For comparison, the cannabinoid content is shown in 



92 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 

aerial material stripped from the stems of 3 week and 1 1 week old plants of the same 
cultivar. Three replicates of each sample were analysed. 



Material analysed 

(n = three analytical samples from 
a single batch) 


CBC % w/w 
Mean ± sd 


THC % w/w 
Mean ± sd 


CBC as % of 
THC+CBC 

Mean ± sd 


25 pm sieve contents 
From 3 week old vegetative plants 


4.25 ± 0.74 


22.70 ± 4.36 


A A C\ O i f\ A C\ 

14.98 ± 0.19 


45 pm sieve contents 
From 3 week old vegetative plants 


6.37 ±0.31 


35.31 ±2.44 


14.92 ± 0.23 


Foliage - 
3 week old vegetative plants 


0.08 ± 0.03 


0.67 ±0.16 


10.43 ±0.89 


Foliage and floral material - 
1 1 week old plants 


0.13 ± 0.01 


8.61 ± 0.45 


1.48 ±0.47 



Table 4.4. The relative proportions of CBC and THC in sessile trichome rich preparations made 
by sieving dislodged trichomes from vegetative foliage of high-THC clone Gl M3. Three 
subsamples of each were analysed by GC 

The CBC potency of the preparation collected on the 45 pm sieve (6.37 % w/w) was 
approximately eighty times greater than that of the plant material from which it was 
derived (0.08 % w/w). However, the preparation collected on the 25 pm sieve was less 
potent (4.25 % w/w). The reason for this was not known. The smaller trichomes would 
have a greater surface area to volume ratio and it is possible that these contained 
proportionally more outer membrane and vesicle-associated fibrillar material. Despite 
the high proportions of THC remaining in these preparations, the results indicated that 
the trichome separation technique does enable samples to be produced which have a 
favourably altered cannabinoid profile. It is notable that the proportion of CBC within 
the cannabinoid profile of the separated trichomes (approximately 15% w/w) was 
greater than that of the foliage (approximately 10% w/w). The proportion fell further as 
the plant matured, repeating the observation reported by de Meijer etal., (2008). 

The reason for the observed difference in proportion of CBC in the trichomes and the 
foliage from which they were collected is not clear. Two possibilities are suggested: - 

1) Although the foliage was collected from three week old plants, many of the 
trichomes residing there would have formed earlier during the plants development. 



93 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



The cannabinoid profile of these trichomes would reflect the proportionally higher CBC 
expected in vegetation that was less than three weeks old. 

2) In the three week old foliage there would be many new sessile trichomes forming. 
Following the well reported ontogenetic pattern, these would have a lower proportion of 
CBC than their predecessors. It is possible that these were not sufficiently resilient to 
be dislodged intact during agitation in iced water. The filtrate would therefore be 
skewed towards more mature trichomes. 

4.5.3 The collection of sessile trichomes from foliage of a high CBC 
chemotype as a means of isolating the minor cannabinoid CBC 

The preparation of sessile trichomes collected from vegetation of clone M240 was dried 
overnight. The material proved an extremely rich source of CBC - 44% w/w. The 
sample also exhibited a high level of purity. As found in the previous test, the fall in the 
proportion of CBC in the cannabinoid profile of the enriched trichome preparation 
appeared to lag behind that of the plant material from which it was collected. The 
foliage, in which CBC accounted for 61% of the total cannabinoids, had produced a 
trichome filtrate within which CBC accounted for 94% of the cannabinoid total (Table 
4.5). Patent protection of this procedure, as a means of producing purer sources of 
CBC, was applied for (GB 0806553.4.). This material was subsequently further 
processed to produce a CBC sample exceeding 99% purity levels, which was 
submitted for in-vitro pharmacological evaluation. 





Cannabinoid % w/w mean ± SD 




Sample 
analysed 


CBC 


CBCV 


CBG 


CBD 


% Purity 
CBC ± SD 


Trichomes 


44.37 ± 4.95 


0.57 ± 0.07 


0.33 ± 0.04 


0.60 ± 0.06 


94.2 ± 0.3 


Foliage 


1.36 ±0.04 




0.62 ± 0.02 


0.25 ±0.01 


61.0 ±0.9 



Table 4.5. The proportions of principal cannabinoids (±SD) found in a trichome-rich filtrate 
clone M240, containing only sessile trichomes. One bulk sample was prepared and four sub- 
samples analysed. Also shown is the purity of the CBC (expressed as a %w/w of total 
cannabinoids detected) within the foliage from which these trichomes were collected. 



4.5.4 Ontogenetic changes in glandular trichome secondary metabolite 
content 

The changing terpene profile of glandular trichomes collected from plants at a range of 
developmental stages is shown in Tables 4.6 (THC chemovar) and 4.7 (CBD 
chemovar). 



94 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



In the high-THC plants harvested 6 to 10 weeks after being placed in short daylength, 
the monoterpenes accounted for between 60%-67% of the monoterpene/sesquiterpene 
total (when assessed by peak area). There was no clear trend against time. The 
trichomes from the young foliage were notable for having a much lower proportion of 
monoterpenes (26%) than those from the flowering material. However, it can be seen 
that not all monoterpenes showed the same contrasting pattern. While the proportion of 
a-pinene, (3-pinene and limonene is similar in both, the proportion of myrcene in the 
profile of foliar sample is much lower. The reason for this is not known. However, it is 
important to note, that in some cases, a range of terpenes can be synthesised with the 
involvement of a single enzyme (Croteau and Johnson, 1984). As a consequence, the 
ratio of the terpenes produced always maintains a fixed ratio, a-pinene, (3-pinene and 
limonene, may be linked in this way while myrcene appears not to be linked to other 
terpenes. 



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Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



Raw material used 


Foliar 


Foliar and Floral Mixture 


Trichome tvDe 

ill \j i i \s ill \-> i y k/ 


Sessile 


Predominantly Stalked 


Wks after planting 


3 


9 


10 


11 


12 


13 


VA/lro in 19 hr rla\/o 
VVKb 111 IZ 111 Udyo 





6 


7 


8 


9 


10 


Monoterpenes 


% Peak Area 


a-pinene 


0.9 


0.7 


0.8 


0.8 


0.9 


1.0 


3-pinene 


1.7 


1.5 


1.7 


1.9 


2.1 


2.2 


Myrcene 


14.8 


47.7 


51.8 


46.8 


44.8 


44.7 


Limonene 


8.5 


8.4 


9.3 


9.1 


9.2 


9.4 


Linalol 




2.5 


3.0 


3.0 


3.1 


3.2 


Sesquiterpenes 


% Peak Area 


trans caryophyllene 


21.1 


18.1 


14.8 


17.4 


17.5 


17.5 


trans a 
bergamotene 




1.9 


1.7 


1.9 


2.0 


1.9 


(z)-(3 farnesene 


9.7 


3.9 


3.3 


3.5 


3.7 


3.5 


a caryophyllene 


16.0 


7.4 


6.4 


7.2 


7.6 


7.3 


(e)-(3 farnesene 


8.6 


1.7 


1.6 


1.8 


2.0 


2.0 


gurjunene 


4.9 


2.0 


1.8 


1.9 


2.1 


2.0 


A guaiene 


2.2 












(e)-nerolidol 


3.3 


4.1 


3.9 


4.6 


5.0 


5.2 


Unidentified 


3.5 


3.1 


3.2 


3.8 


4.3 


4.6 


Unidentified 


1.8 


3.1 


2.8 


3.0 


2.9 


2.7 


a-bisabolol 


2.9 












Monoterpenes 


25.9 


60.9 


66.6 


61.6 


60.1 


60.6 


Sesquiterpenes 


74.1 


39.1 


33.4 


38.4 


39.9 


39.4 



Table 4.6. The terpene profile of essential oils produced by steam distillation of glandular 
trichomes extracted from high-THC clone G2 M6 at various stages in the plant's development. 
The results are the relative peak area after analysis of the essential oil by GC. Missing values 
occur where individual terpene contents were below the detectable limits. 



96 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



Raw material 
used 


Foliar 
i uiicii 


Foliar and Floral Mixture 


Trichome type 


Sessile 


Predominantly Glandular Stalked 


Wks after 
nlantinn 

UIQI 1 LI 1 i y 


3 


9 


10 


11 


12 


13 


Wppk<; in flnwpr 





6 


7 


8 


9 


10 


Mnnntprnpnp^ 

IVIL/I IULGI UCI ICO 


% Peak Area 


n-ninpnp 

\A VJII Id IU 




9.1 


8.0 


6.8 


8.0 


6.7 


R.ninpnp 
yj l/ii ici ic 




3.7 


3.1 


3.3 


3.6 


3.1 


Myrcene 


16.0 


41.8 


39.7 


50.3 


49.1 


51.1 


Limonene 




4.6 


4.6 


6.0 


5.9 


6.3 


P-ocimene 


3.0 


9.0 


8.4 


11.0 


9.9 


10.3 


Sesquiterpenes 


% Peak Area 


Trans 
rarvnnhvllpnp 

\jCa I y i^l^i ly nci It? 


49.8 


22.5 


25.6 


15.1 


15.9 


14.4 


n ppr\/nnh\/llpnp 

u KjO I y^L^i ly lid ic 


3.2 


5.9 


6.8 


4.2 


4.4 


4.2 


(e)-(3 farnesene 


12.4 


2.4 


2.7 


2.3 


2.2 


2.6 


Unidentified 


0.6 












Unidentified 


6.2 












(e)-nerolidol 


6.3 


1.0 


1.1 


1.1 


1.1 


1.2 


Caryophyllene 
Ox 


0.7 












a bisobolol 


1.7 












Monoterpenes 


19.1 


68.2 


63.8 


77.4 


76.4 


77.6 


Sesquiterpenes 


80.9 


31.8 


36.2 


22.6 


23.6 


22.4 



Table 4.7. The terpene profile of essential oils produced by steam distillation of glandular 
trichomes extracted from high-CBD clone G5 M13 at various stages in the plants development. 
The results are the relative peak area after analysis of the essential oil by GC. Missing values 
occur where individual terpene contents were below the detectable limits. 

Unfortunately, in the CBD-chemovar, the a-pinene, P-pinene and limonene 
concentrations were below the detectable minimum in the foliar fraction and a similar 
comparison was not possible. In this chemovar monoterpenes accounted for 64-68% of 
the terpene total in the first two harvests. However, this rose to 76-78% and remained 
stable for the last three weeks when plant development ceased. As with the high-THC 
clone, the proportion of monoterpenes was much lower in the young vegetative foliage 
(19%). It appears likely that the slightly higher proportion of sesquiterpenes in the 



97 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



younger flowering plants was due to these having a higher proportion of sesquiterpene- 
rich leaves. The proportion of foliage would have fallen as floral development 
continued up to the eighth week after induction of flowering. The higher proportion of 
sesquiterpenes in less developed plants observed here mirrors a similar observation 
reported when a hemp crop was grown outdoors for the commercial production of 
essential oils (Meier and Mediavilla, 1998). A detailed evaluation of terpene production 
in trichomes in field-grown plants is described later in Chapter 6. 

Insufficient entriched trichome preparation was available to enable analysis of the 
cannabinoid content. However it was possible to assess the cannabinoid profile of the 
original plant material. The results are shown in Table 4.8 and 4.9. 



Weeks after planting 


3 


9 


10 


11 


12 


13 


Weeks in flower 




6 


7 


8 


9 


10 


Cannabinoid 


%w/w 


THC 


0.40 


7.92 


8.94 


8.92 


8.92 


8.33 


CBC 


0.06 


0.14 


0.13 


0.12 


0.12 


0.11 


CBG 




0.11 


0.11 


0.12 


0.13 


0.11 



Table 4.8. The cannabinoid profile of the original plant material (high-THC clone G2 M6) from 
which the trichome rich preparations were made. Six subsamples were combined and milled to 
produce one sample for analysis by GC. A missing value denotes that the cannabinoid level was 
below the detectable threshold. 



Weeks after planting 


3 


9 


10 


11 


12 


13 


Weeks in flower 




6 


7 


8 


9 


10 


Cannabinoid 


%w/w 


CBD 


0.20 


2.93 


3.14 


3.10 


3.73 


3.16 


CBC 




0.16 


0.16 


0.14 


0.16 


0.13 


THC 




0.20 


0.17 


0.15 


0.16 


0.14 


CBG 




0.03 


0.03 


0.02 


0.03 


0.02 



Table 4.9. The cannabinoid profile of the original plant material (high-CBD clone G5 M13) 
from which the trichome rich preparations were made. Six subsamples were combined and 
milled to produce one sample for analysis by GC. A missing value denotes that cannabinoid 
level was below the detectable threshold. 

Plant development was seen to have ceased by their eighth week in short daylength, 
and plants harvested after nine and ten weeks showed very high levels of foliar 



98 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



senescence. The results of the cannabinoid assay suggest that both cultivars had 
reached maximum potency at the end of the seventh week in short daylength. In both 
chemotypes, delaying the harvest beyond the eigth week of flowering appears to have 
no clear effect on the terpene or cannabinoid profiles. The foliage from three week old 
vegetative plants showed very low levels of cannabinoid and terpene, as expected. 

The remarkably unchanging cannabinoid and terpene profiles, from the eighth week of 
the flowering period onward, suggest that the secondary metabolites are stable while 
sequestered in the trichome. This facilitates the task of the grower of pharmaceutical- 
grade cannabis, as delaying harvest does not appear to affect the specified ratio of 
metabolites. This is in contrast to observation in other species where the terpene profile 
changes due to enzymic conversion of one terpene to another. In peppermint Mentha 
piperita L. for example, menthone is converted to menthol in the latter stages of 
flowering. (Gershenzon, 2000) Research also showed that catabolism of 
monoterpenes occurs in intact glandular trichomes of mint (Croteau and Martinkus, 
1979). 

The much higher secondary metabolite content of floral tissue compared to the foliage 
is typical of most plant species. The loss of floral tissues is likely to have greater 
impact on a plant's ability to pass on its genes to the next generation. The Optimal 
Defence Theory suggests that, away from the influence of man, plants will have 
evolved to generally allocate secondary metabolites to tissues in direct proportion to 
their value. (Herms and Matson, 1992). The very different balance of monoterpenes in 
the sessile trichomes on the foliage and the predominantly capitate stalked trichomes 
on floral tissues is supporting evidence that these trichomes have different functions. 
Both types contain bitter sesquiterpenes which can act as anti-feedant repellents. The 
increased monoterpene content of capitate stalked trichomes would be expected to 
lower the viscosity of the contents, thereby making it more able for them to ensnare 
insects, as reported in the previous chapter. The monoterpenes are more volatile, and 
being hydrophobic they are highly persistent in the atmosphere. Insect olfactory 
systems are devoid of the mucous membranes found in mammals, and they are 
especially sensitive to such lypophylic chemicals. Monoterpenes are thereby detected 
by insects at considerable distances from the plant. In many cases these 
monoterpenes are repellent to insects, (e.g. a-pinene and ants) those insects 
apparently misidentifying the monoterpene as an alarm pheromone (Kelsey et al., 
1984). It is notable that the secondary metabolite profiles of both chemotypes (Tables 
4.6 and 4.7) were dominated by the most reduced forms of terpenes - the 'true 
terpenes' - containing just carbon and hydrogen. These were the most costly type to 



99 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



biosynthesise (Gershenzon, 1994). The slightly more oxidised monoterpene alcohols 
(e.g. linalool,) are more water soluble (Merck Index, 1996) and less persistent in an 
atmosphere containing water vapour. It is possible that it is a function of the 
cannabinoids, by virtue of their anti-oxidant properties, is to prevent oxidation of the 
monoterpenes to more soluble monoterpene alcohols. 

Some of the high monoterpene content of glandular trichomes is possibly due to 
human influence over many centuries. The highest monoterpene contents are most 
likely found in plants producing high quantities of resin. These plants would have been 
selectively planted by growers striving for maximum resin yields. Many would also find 
the odour attractive and of greater demand for those using fragrant resins as religious 
sacraments. Focused plant breeding of cannabis for pharmaceutical use continues to 
make dramatic changes to the glandular trichome contents. 

4.6 CONCLUSIONS 

The previous chapter showed that on mature flowering plants, only a very small 
proportion of the total cannabinoid content is sequestered in sessile trichomes, the 
majority being synthesized in the capitate stalked form. On a mature flowering plant, 
the cannabinoid content of the sessile trichomes has an almost negligible influence on 
the total cannabinoid content. Comparisons of proximal and distal bract sections, with 
differing ratios of sessile and capitate stalked trichomes, suggested however that the 
former had a higher CBC content within the cannabinoid profile. By separating and 
then analysing sessile and capitate stalked trichomes, the research reported in this 
chapter confirmed this to be the case. 

Although yields were comparatively small, enriched trichome preparations could be 
produced from foliage, within which only the sessile form was present. Agitating the 
foliage in cold water (<1°C) appeared to dislodge a higher proportion of the more 
mature sessile trichomes, within which there was a higher proportion of CBC than that 
of the overall sessile trichome population. Using the foliage of a chemovar, within which 
CBC accounted for approximately 61% w/w of the cannabinoid total, an enriched 
trichome preparation could be produced, within which CBC constituted 94% w/w of the 
cannabinoid total. The CBC content of the dried preparation was 44% w/w. This was 
possibly the first time that such a rich source of CBC had been extracted from cannabis 
material by such simple means. 

In marked contrast to some other species, this chapter showed that in Cannabis the 
terpenoid content of trichomes appears to be stable at the end of the flowering period, 
at least in the glasshouse growing environment. The cannabinoid and terpene profile 



100 



Chapter 4. The Function and Exploitation of Secondary Metabolites from Glandular Trichomes of Cannabis sativa L. 



of the phytopharmaceutical feedstock would be minimally affected by a few days 
alteration to the intended harvest date. This indicates that some flexibility in harvest 
timing is justified. 

Capitate stalked trichomes appear to have a higher monoterpene:sesquiterpene ratio 
than the sessile type. Although only the sessile trichome form is present on foliage, 
floral material predominantly exhibits the capitate stalked form. As a consequence of 
this marked difference if trichome distribution, floral material has a higher 
monoterpene:sesquiterpene ratio than foliage. Any alteration to growing conditions or 
harvest timing, which affected the ratio of foliar and floral material, would be likely to 
affect the balance of mono and sesquiterpenes in the feedstock produced. 

The previous chapter showed that only the capitate stalked trichome form was involved 
in insect entrapment. The higher proportion in monoterpenes in this form implies that 
the secretory head contents would consequently be of lower viscosity than those of the 
sessile form. If punctured by insect contact, the contents would more readily spread 
over the insect surface. As the monoterpenes from the punctured trichome volatalised, 
this would leave an increasingly viscous adhesive coating on the insect. Microscopic 
observations indeed showed the capitate trichome contents to have extremely 
adhesive qualities. 

The following chapter evaluates glasshouse growing methods with the aim of enabling 
the reliable and uniform propagation of high yielding phytopharmaceutical feedstocks, 
with a high density of capitate stalked trichomes. Uniform foliage:flower ratios would 
appear to be vital if both materials are included in the feedstock. 



101 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



Chapter 5 Indoor Propagation of Medicinal Cannabis 

5.1 INTRODUCTION 

To enable year-round propagation of high yielding good quality plant material for 
GW Pharmaceuticals, extensive research was needed to identify suitable horticultural 
methods. The techniques initially adopted were recommended by consultants from 
HortaPharm BV. Additional advice was sought from the books on cannabis horticulture 
then available from at least a dozen authors (e.g. Clarke, 1981 ; Frank, 1997; Rosenthal, 
1998). Although the first of these books sourced the work of both scientific and the 
clandestine growers, most publications appeared to rely totally on the latter and 
contained minimal input and peer review from named qualified and respected 
scientists. Indeed the growing of cannabis was often described as more of an art than a 
science. Within three weeks of planting the first seed, insect damage was observed on 
the crop and the battle against pest and disease had begun. Growth media had to be 
developed that supported good root development, and fertilizer regimes had to be 
devised to give the correct balance of nutrients at the appropriate time in the plants' 
development. As the results of these studies are also of interest to illicit cannabis 
growers, this aspect of the research cannot be included in this thesis. 

Along with the three most commonly used legal drugs in the west - alcohol, nicotine 
and caffeine - cannabis-derived THC is unusual, if not unique, amongst illicit drugs in 
that part of the enjoyment for many users is the taste and odour of the plant material 
from which it is sourced. In addition to raising yield and potency, many of the cannabis 
growing techniques for illicit cannabis were designed to improve the taste of the 
harvested material. This would be of little or no relevance when growing cannabis as a 
phytopharmaceutical, and this typifies how the aims of the illicit and pharmaceutical 
growers differed. The research discussed in this thesis also differs in that it investigated 
the effect of growing conditions on the biosynthesis of cannabinoids other than just 
THC. To enable the tight specification expected of a phytopharmaceutical feedstock to 
be met, the research reported here placed greater emphasis on crop uniformity than 
crop yield. There was little doubt that environmental conditions influenced the quantity 
of cannabinoids in various plant parts at different growth stages, as demonstrated by 
Fairbairn (1976), Lydon and Teramura (1987) and others. An improved knowledge of 
how alterations to the growing method affected the secondary metabolite profile would 
reduce the likelihood of unacceptable feedstock being produced. It would also increase 
the ability to perform a reactive diagnosis, if a batch of feedstock was produced that 



102 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



was 'out of specification'. Reviewing the findings of several researchers, Raman and 
Joshi (1998) highlighted the magnitude of this challenge. They reported that even if 
genetic and environmental factors could be tightly controlled, high interplant variability 
in cannabinoid content could be expected between plants of the same chemotype 
when grown under identical conditions. 

Initial advice was that plants should be propagated from cuttings rather than seed. 
Most researchers agreed that genetics exert the greatest control of a plant's 
cannabinoid profile (Beutler and Der Manderosian, 1978; Fournier et al., 1987; 
deMeijer, 1994). The propagation of cannabis from cuttings guaranteed the genetic 
uniformity of all plants produced from the same source (Samuelsson, 1999). 
Propagation from seed however was perceived to be less labour intensive, and with 
appropriate research perhaps the foreseen variation could be dispelled or overcome. 
Research reported here compared the variability of cloned and seed-sown plants. 

To produce a medicine containing a desired even balance of cannabinoids, separate 
THC and CBD chemotypes were routinely propagated for Sativex® production. By 
blending Botanical Drug Substances from these two, it could be guaranteed that the 
correct ratio of these cannabinoids would be delivered. It has been concluded 
elsewhere that the inheritance of CBD and THC chemotype was controlled by a 
monogenic, co-dominant mechanism (de Meijer et al., 2003). A single locus referred to 
as B was postulated, with two alleles, 6 D and S T , encoding CBD- and THC synthase 
respectively. According to this model, the CBD chemotype had a B D /B D genotype at the 
B locus, and the THC predominant plant had a S T /6 T genotype. Heterozygous B D /Bj 
genotypes were available that produced both cannabinoids, thereby possibly avoiding 
the need to blend materials. Research reported here was performed to assess how 
growing methods and harvest timing affected the ratio of these two cannabinoids in 
Bq/ Bj genotypes. 

Prior to research for this thesis, large seasonal variations were seen in plant yield. In 
winter crop yields fell by two-thirds and the fall in cannabinoid yield was even more 
pronounced. In addition to the economic implications, this variability in potency may 
have also unacceptably affected the secondary metabolite profile of the Botanical Drug 
Substance (purified extract) made from that feedstock. While glasshouse temperature 
remained constant throughout the year, summer light levels far exceeded those in 
winter, and this was the suspected main cause of the yield variation. Limited growth 
room tests, performed in winter, showed that when summer irradiance levels were 
recreated, both plant yield and cannabinoid assay values returned to those typical of a 
summer crop. Extensive written guidance was available regarding the use of lighting 



103 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



systems to support cannabis growth. Most was aimed at those growing under totally 
artificial lighting conditions and few described the use of electrical lighting to 
supplement natural daylight. Growing cannabis this way in a glasshouse had been and 
continues to be less common, at least in the UK (ACMD, 2008), partly due to it being 
too difficult to keep the activity secret and secure. The effect of lighting on cannabis 
growth was discussed in little detail in peer-reviewed scientific literature. 

It was well recognised that the floral material of the female plant was the most potent 
source of cannabinoids (Fairbairn, 1976). To confirm this, various parts of some 
unpollinated THC chemovar plants were analysed. The THC content of dried seeds, 
roots, stems, leaves and inflorescences was found to be 0.0%, 0.0%, 0.3%, 0.8% and 
15.2% w/w respectively. When the outer bracts and stalks were removed the THC 
content of the remaining inflorescence material was found to be over 18% (Potter 
2004). In a separate test pollination was found to reduce THC content by more than 
half, with THC yield per unit area being decreased by over 75%. It was therefore 
essential to grow plants though to the flowering stage to obtain the highest yielding 
material. 

Cannabis is generally a 'short day plant' that by definition only commences to flower 
late in summer, once the day length starts to fall (Clark, 1981). When the so called 
critical daylength is reached floral development is stimulated. More correctly, the plant 
is responding to the increasing length of the night, during which time a light-sensitive 
phytochrome protein slowly dimerises to a different form. Within seconds of light 
exposure the protein reverts back to the original structure. It is only when the night time 
is sufficiently long that a required balance of the dimers is reached to signal 
commencement of flowering (Halliday and Fankauser, 2003). To reliably induce 
flowering in most varieties of cannabis, the night-length must be greater than the critical 
value. The critical daylength for an individual variety is greatly affected by its 
geographical origin and would generally be greatest in those plants derived well away 
from the equator (de Meijer and Keizer, 1994). Exceptions to this response occur in 
plants adapted to grow in equatorial regions, where there is minimal variation in day 
length. Flowering in tropical cannabis plants is more closely related to plant age. In 
contrast, rapid flowering ecotypes are found at latitudes of 60 e (Callaway, 2002) or 
more. These have typically adapted to survive in the very short growing season, and 
commence flowering early in the season irrespective of the daylength. Despite these 
differences in critical daylength, it has been common practise amongst illicit cannabis 
indoor-growers to induce plants to flower by placing them in a twelve hour daylength. 
The use of such a short day length to induce flowering was perceived as somewhat 



104 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



ironic, as in the northern hemisphere an autumn day length of twelve hours occurs 
naturally at the equinox during the last days of September, when cannabis flowering 
would be finishing. A slightly longer day length would still initiate flowering and would 
result in proportionally more light energy being delivered to the plant in one day. 
Research reported here investigates how plant development, yield and cannabinoid 
profile are affected by differing daylengths of eleven, twelve and thirteen hours. 

Recreational cannabis users would often harvest their plants when about 95% of the 
visible stigmas had senesced, but this would vary according to the variety and the 
grower's personal preference (Clarke, 1993). This was partly related to taste of the 
material as well as its potency. It was not known how closely inflorescence 
development stage was related to the secondary metabolite content. It was predicted 
that the length of the propagation period would not greatly affect the cannabinoid 
profile, especially of those cannabinoids that are the final products of separate 
biosynthetic pathways. Rowan and Fairbairn (1977) and others had shown that during 
the flowering phase CBD chemotypes would produce a small proportion of THC and 
THC chemotypes would produce a small proportion of CBD. In THC chemotypes, 
although substantial proportions of CBC were often present in the early stages of 
growth, THC dominated the profile of THC chemotypes throughout the flowering stage. 
However, how the state of maturity of the plant affected the relative proportions the 
precursor CBG and the final cannabinoids was less well studied, and was investigated 
in this study. Based on visual assessment, clones of the THC chemotype typically 
appeared ready for harvest eight weeks after being placed in a twelve hour day length. 
The objective of this investigation was to check that this flowering time was the 
optimum to achieve efficient production of THC. The investigation would also 
investigate how the duration of the flowering period affected the relative proportions of 
THC and its precursor CBG. 

The methods of production of licensed medicines had to comply with Rules and 
Guidance for Pharmaceutical Manufacturers and Distributors 2007 as directed by the 
Medicines and Healthcare Products Regulatory Agency (MHRA, 2007) to ensure that 
they were of sufficient quality. It was perceived that GMP did not apply to the 
propagation phase of the production process, but did apply once the plant material was 
processed. Although the cannabis plants were to be grown in a glasshouse, the 
research described in this chapter was performed in the knowledge that the 
propagation phase would have to comply with the EMEA Good Agricultural Practice 
(GAP) Guidelines (EMEA, 2006). The use of the word agriculture in this quality control 



105 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



system reflects that most, if not all of the existing phytopharmaceuticals in western 
medicines, were derived from outdoor grown materials. 

5.2 AIM AND OBJECTIVES 

A series of tests was performed, all of which had the aim of identifying how glasshouse 
growing methods could be altered to maximise plant quality and uniformity. The linked 
objectives were as follows: 

5.2.1 To gain a greater understanding of the irradiance levels on plant yield and 
cannabinoid content. 

5.2.2 To confirm or dispel the belief that plants propagated from clones were 
significantly more uniform in cannabinoid profile than plants grown from seed. 

5.2.3 To ascertain how the length of flowering period affected the cannabinoid yield. 

5.2.4 To ascertain how the length of flowering period affected the cannabinoid 
profile of THC chemovars and heterozygous plants with a mixed THC/CBD 
profile. 

5.2.5 To compare the effect of 13, 12 and 1 1 hour daylengths on plant development, 

cannabinoid yield, potency and profile. 



106 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



5.3 MATERIALS 

5.3. 1 Plant Propagation and Drying Materials 



Materials 


Source 


Complete light fittings incorporating 600 watt Osram 
Nav T High Pressure Sodium Lamps, compatible 
ballasts and Silver Wing® reflectors. 


Horti systems UK Ltd 
Pulborough, UK 


Complete light fittings incorporating 600 watt Hortilux- 

■ i i' i r~\ t~\ i" i i ■ i ■ 

Scroeder High Pressure Sodium Lamps, compatible 
ballasts and Deep reflectors 


1000 watt Mercury Vapour MBFU lamps 


Seradix® Rooting Powder (MAPP No 1331) 0.3% w/w 
4-lndol-3yl butyric acid 


Certis UK 1 b Mills Way, 
Amesbury, UK 


Jiffv 7Rootina Pluas 

\J IIIV / 11 V-/ LI 1 1 1 1 \mA \A \j 


Jiffy Products International 
AS, Kristiansand, Norway 


i nnpex-rius sacneis /\mDiys6ius cucurncris Tor 
biocontrol of Thrip Thrips tabaci 


Koppert B.V. Rodenrijs, 
NL 


Spidex Phytoseiulus persimilis for biocontrol of red 
spider mite Tetranychus urticae 


En-strip Encarsia formosa for biocontrol of white-fly 
Trialeurodes vaporariorum 


Aphipar bottles Aphidius colemani for biocontrol of 
cotton-melon aphid Aphis gossypii 


Ebac BD150 commercial building drier 


Ebac Ltd., Durham, UK 



Table 5.1. Plant Propagation Materials 



107 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



5.3.2 Germplasm Details 



GW 
Accession 
Number 


Variety/ 
Clone 

Name 


Supplier 


G1-M1 
GP-M7 

\J L— 1 VI / 

G5- M11 
G5- M13 
G5- M16 


Gwen 

\_3M id 

Gayle 
Grace 
Gill 


Seed from HortaPharm BV Schimkelhavemkade 1075 VS 
Amsterdam, NL, used to produce clones by 

GW Pharmaceuticals Ltd, Porton Down Science Park, 
Salisbury, Wiltshire 


M237 




Clone bred by GW Pharmaceuticals (As above) 






Various suppliers as follows: 


G41 to 
G52, G63, 
G91 G130 
and G159 


Various 


Sensi Seeds B.V., Rotterdam, NL 
Natural Mystic, Rochdale, UK 
Nirvana, Zaandam, NL 
Dutch Passion B.V. Amsterdam, NL 
Serious Seeds, Amsterdam, NL 
Seedsman Ltd, London, UK 



Table 5.2. Germplasm Details 



5.3.3 Light Measurement and Weighing Equipment 



Skye 500 Hand-held Light Sensor 


Skye Instruments, Powys, UK 


Kipp and Zonnen solarimeter 


Priva UK Ltd, Gloucestershire, UK 


Salter M323 Top Pan Balance 


E&G Websales Ltd, Delfryn, Lixwm, 
Holywell, UK 



Table 5.3. Light Measurement and Weighing Equipment 



5.3.4 Growth Medium 

The growth medium used for these studies was developed at GW Pharmaceuticals Ltd, 
Porton Down Science Park, Salisbury, Wiltshire and its precise formula has to remain 
proprietary information (Wilkinson 2006). The medium (or compost) primarily consisted 
of peat and perlite and included sufficient nutrient to maintain healthy plant growth 
through to harvest with no additional feeding. 



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5.4 METHODS 

Before describing the more detailed experimental methods that apply specifically to 
individual studies, sections 5.4.1.1 to 5.4.1.11 describe the routine propagation and 
botanical raw material production processes which were routinely used and applied to 
most of the studies described in this chapter. Sections 5.4.2.1 to 5.4.2.11 describe 
more specific methods applying to individual tests within this chapter. 

5.4. 1 Routine Propagation and Plant Production Methods 

5.4.1.1 Seed sowing and transplantation of seedlings 

Seeds were sown in trays of a range of proprietary seed compost. There is no light 
requirement for germination and seeds were sown approximately 1 cm deep and at 
least 1cm apart. The compost was sufficiently deep (> 3 cm) to allow some natural 
downward root development prior to replanting. The compost was lightly compressed 
after sowing, and well watered. The temperature was maintained at 20-25°C and 
lighting adjusted to maintain a 24 hour daylength. Horticultural fleece was placed over 
the seed tray or pot until seedling emergence to reduce surface drying of the compost. 

In ideal conditions seedlings were ready for transplanting approximately ten days after 
sowing, by which time the hypocotyls were typically around 10 cm tall and the first pair 
of true leaves well formed. Seedlings were teased from the seed compost and 
transplanted into individual pots of the appropriate growth medium. A sufficiently deep 
hole is prepared in this medium to allow the seedling to be lowered undamaged so that 
the first pair of true leaves sat 1-2 cm above the surface of the medium. The 
surrounding growth medium was firmed by hand, and the growth medium sufficiently 
watered to maintain moisture around the seedling. 

5.4.1.2 Production of Cuttings (Clones) 

Branches of vegetative material were removed from plants producing ample numbers 
of axial buds. (Figures 5.1 a-f). The branch was then cut into sections, each carrying 
one axial bud and retaining approximately 5cm of stem below the bud. The stem below 
the bud was dipped in rooting powder containing 0.3% w/w 4-lndol-3yl butyric acid and 
then promptly into a very moist peat plug. These plugs were placed on a bench of 
regularly wetted gravel under a polythene cover to maintain very high humidity. 
Irradiance levels under the polythene was maintained at approximately 10 - 15 W m" 2 
for 24 hours per day. Successful cuttings were normally producing sufficient roots 
within fourteen days to enable repotting. 



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5.4. 1.3 Nurturing Vegetative Growth of Seedlings and Cuttings. 

For the first three weeks vegetative growth was encouraged by maintaining the 
cannabis plants in a twenty-four hour daylength. This supported maximum growth rate 
by enabling continuous photosynthesis. Being 'short-day plants', flowering was 
naturally prevented in this regime. 

Unless otherwise stated, all high-THC plants are potted in five litre pots of GW 
Pharmaceuticals' standardised peat-based growth medium. High CBD plants of variety 
G5 are potted in three litre pots of the same medium. These were closely placed pot-to- 
pot on the glasshouse or growth-room bench for three weeks before being induced to 
flower. A proportion of plants were retained in long daylength however to produce 
ample vegetative material for the production of cuttings. 

5.4.1.4 Induction and Maintenance of Flowering 

Plants were relocated, or the lighting regime adjusted, so that plants were now in a 
short daylength. Unless otherwise specified, this was twelve hours per day. The 
response to daylength change was for plants to commence flowering within seven to 
fourteen days. The short daylength was maintained for the entire flowering period, 
which typically lasts eight weeks. During this period, unless otherwise specified, crops 
were spaced at a density of 10 m" 2 (THC chemotype) or 17 m" 2 (CBD chemotype). 

5.4.1.5 Biological Pest Control 

The major pests encountered were red spider mites {Tetranychus urticae), the onion 
thrip (Thrips tabaci), white fly {Trialeurodes vaporariorum) and cotton-melon aphid 
(Aphis gossypii). These were controlled by regular introduction of the predatory mite 
and insect species Phytoseilius persimilis, Amblyseius cucumeris, Encarsia formosa 
and Aphidius colemani (Malais and Ravensberg, 1992). 

5.4.1.6 Harvesting 

Plants were harvested by cutting the stems just below the lowest side-branch and 
placed on a clean surface if being temporarily stored prior to drying. As respiration 
could rapidly generate heat within piled fresh material, drying of harvested plants was 
commenced as soon as possible to avoid heat-induced catabolism. 



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Figure 5.1. Production of cannabis cuttings. A vegetative cannabis branch (a) is cut into 
sections (b). Each cutting has been cut leaving approximately five centimetre of stem below a 
single axial bud and up to one centimetre above (c). The base of the cutting is dipped in rooting 
powder (d) and then placed in moist peat plugs (e). After two weeks roots are protruding from 
the peat plugs and the cutting is ready to be planted. 



5.4.1.7 Crop Drying 

Plants were hung to dry in a closed environment and industrial dehumidifiers used to 
lower air moisture (Figure 5.2). Horticultural fans were used to maintain air circulation. 
Freshly harvested material had an aqueous content of approximately 80% (w/w). The 
air surrounding the chamber was conditioned as appropriate to ensure that waste heat 
from the dehumidifiers did not raise temperatures within the chamber above 35°C. The 



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crop was considered dry enough for storage or processing when below 15% (w/w). At 
his point, the crop was clearly crisp to touch, the inflorescence tissue closest to the 
stem feeling dry and the floral material would readily pull away from the stem without 
excessive force. 



Figure 5.2. A newly harvested crop hung to dry on wires. 

5.4.1.8 Stripping 

When sufficiently dry, all floral and leaf material was stripped from the stem by pulling 
the plant though the tightly clasped thumb and forefinger of a gloved hand. This floral 
and foliar mixture is referred to as Botanical Raw Material (BRM). 

5.4.1.9 Garbling 

Tyler et al., 1998 defined garbling as "the final step in the preparation of a crude drug 
and involving the removal of extraneous matter such as other parts of the plant, dirt 
and added adulterants. This is done to some extent during collection and should be 
carried out after the drug is dried and before it is baled and packaged". 

When grown in the glasshouse, minimal adulteration or contamination with dirt arose. 
The presence of low-potency stem material was undesirable and all material assessed 
as more than 2 mm in diameter would be manually removed. 

5.4.1.10 Storage 

As with other dried herbs, e.g. hops (Henderson, 1973) the aqueous content of stored 
dried herbs is greatly affected by the surrounding humidity. To minimise the possible 
effects of varying moisture content on microflora activity, secondary metabolite 
volatilisation and catabolism the herbal material was stored in a controlled environment 




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Chapter 5. Indoor Propagation of Medicinal Cannabis 



at 35 ± 5% RH. Low air moisture was achieved using industrial dehumidifiers. THC 
degrades more rapidly in the presence of light (Fairbairn, 1976). To discourage this, 
the material was stored in the dark prior to analysis or use. 

5.4.1.11 Environmental Control System 

Glasshouse temperatures and lighting conditions were controlled by a Priva Universal 
Computer (907) system. Warmed or chilled air was supplied via a rapidly responding 
air handling system to ensure that a target glasshouse temperature was achieved +/- 
1.5°C. Supplementary lighting was programmed to be turned on at the desired times 
of the day, and only when natural daylight irradiance levels were below a selected 
minimum level. Similarly, glasshouse roof shades and or blinds were automatically 
opened or closed to control the ingress of natural light and to prevent light pollution 
from glasshouse lights after sunset. 

5.4.2 Specific Methods 

5.4.2. 1 Uniformity of Plants Grown from Cuttings or Seed 

THC variety G1 was selected for this test. Thirty plants raised from cuttings were 
compared to a similar number raised from seeds. The clones were all derived from one 
plant and code named clone M1. For the seed sown crop, seedlings were raised as 
described in paragraph 5.4.1.1. For the plants raised from cuttings, the propagation 
timings and conditions were as described in paragraph 5.4.1.2. Ten days after sowing, 
eighty individual seedlings were transplanted into pots using identical materials and 
methods employed for the propagation of plants from cuttings. The rooted cuttings 
were also transplanted on the same day. 

Three weeks after transplanting, the plants were moved to a twelve hour light / twelve 
hour dark regime to induce flowering. All male plants and excess female plants were 
removed to leave thirty seed and thirty clone derived plants. These were maintained in 
neighbouring blocks at a density of ten plants per square metre. Eight weeks after the 
move to twelve hour days, the plants were harvested and dried. The total weight of 
foliar and floral material produced by each plant after removal of the stem was 
recorded. This mixture was then milled and the cannabinoid content studied using gas 
chromatography. 



5.4.2.2 Effect of Duration of Flowering Period on Yield 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



Two clones used for the production of clinical trials raw material were selected viz. M1 
and M7. Twelve commercially available varieties were also selected. All were 
commonly used for illicit recreational purposes and were described as being derived 
from tropical or subtropical areas. Plants truly derived from these contrasting locations 
would be expected to differ in the natural duration of their flowering period. 

Seedlings of each of the twelve commercial varieties were raised in the glasshouse in a 
twenty-four hour daylength. When sufficiently developed, cuttings were taken from 
each plant and encouraged to root using the standard method (section 5.4.2). The 
original plants, from which the cuttings were taken, were moved to a twelve-hour 
daylength regime to encourage flowering. All plants consequently identified as male 
were disposed of along with all cuttings taken from them prior to gender identification. 
Up to three female clone lines of each variety were retained for further evaluation, each 
being regarded as the most potent or prolifically flowering examples of their variety. 
Just one clone line was chosen for evaluation from four of the varieties. Two or three 
clone lines were selected from the remainder. In the latter case, these varieties had 
shown a high level of phenotypic variability. 

The propagation regime was based on that used for regular production of THC for 
clinical trial (Section 5.4.2 - 5.4.8). However, a temporary installation of supplementary 
glasshouse lights was in place for this test and this gave a slightly lower minimum 
irradiance level of 40 W m" 2 PAR. Fifteen plants of each clone were propagated and 
five of each harvested after six, eight and ten weeks in flower. Harvested plants were 
dried in a dehumidified environment (final humidity < 35%) for seven days and the floral 
and foliar material stripped from the stem. The floral and foliar material of the five 
replicate plants of each clone line were thoroughly mixed. Five small samples of 
approximately 1 g were taken at random from the mixture, blended and analysed by 
High Performance Liquid Chromatography (Appendix 1). 

5.4.2.3 Effect of Daylength on Cannabinoid Profile (Part 1) Comparison of 12 and 13 
hour daylength 

All plants were propagated in the glasshouse using the standard materials and 
methods. Eighty plants of each selected clone line were grown in five-litre pots and 
maintained in constant twenty-four hour daylength for the first three weeks after potting. 
Thereafter half were transferred to a glasshouse area with a twelve-hour daylength and 
the remainder relocated to a similar glasshouse area with a thirteen-hour daylength 
regime. In both areas the glasshouse target irradiance level was 75 Wm" 2 . Within each 
regime, each clone line was divided into two batches of twenty plants. Single plant 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



batches of each clone line were placed alongside each other at a pot-density of 10 m" 2 . 
Plants were watered by hand throughout the test. Eight weeks after the move to short 
daylength, one batch of each clone was harvested and hung to dry. The remaining 
batches were harvested and dried fourteen days later. 

5.4.2.4 Effect of Daylength on Cannabinoid Profile (Part 2) Comparison of 11 and 12 
hour daylength 

The above method was duplicated. However, this test was located in a growth room 
with no natural lighting (Figure 5.3), and twenty plants of each clone (rather than five, 
as used in the previous test) were included in each replicate. High-pressure sodium 
lamps gave a uniform irradiance level of 70 W m~ 2 . Temperature was maintained at 25 
± 1 °C. 'Ebb-and-flood' benches provided uniform levels of water to all plants. Vertical 
blinds between areas enabled plants to be divided into areas with equal environmental 
conditions but altered daylength. 




Figure 5.3. An experiment to compare plant development and cannabinoid content when 
flowered in 11 and 12 hour daylengths. Plants are maintained on ebb-and-flood benches and 
lighting provided by high pressure sodium lamps. Duplicate batches of plants are maintained 
either side of the curtain with plants on the right receiving the longer daylength regime. 

5.4.2.5 Plant height assessment 

The height of each single plant (from the surface of the pot to the top of the tallest 
inflorescence) was measured by hand immediately before harvest. 

5.4.2.6 Stigma senescence assessment 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



The stage of inflorescence development on each clone was recorded weekly, up to the 
final harvest date, by making a visual estimate of the relative proportions of still-viable 
white stigmas and senesced non-viable brown stigmas (Figure 5.4). 




Newly formed white stigmas are 
viable and receptive to pollen. 



Aged stigmas have senesced to 
a brown colour and are no 
longer viable. 



Figure 5.4. A close-up view of part of an unpolllinated cannabis inflorescence, showing viable 
and older non-viable stigmas. 

5.4.2. 7 Plant Weight Assessment 

After being dried for seven days, the foliar and floral material from each individual plant 
was stripped from the stem and the latter discarded. The weight of combined foliar and 
floral material was weighed on a top pan balance. 

5.4.2.8 Cannabinoid Content and Profile 

The cannabinoid content and profile of the samples from the twelve versus thirteen 
hour assessment were analysed by HPLC. This facility was not available for the eleven 
versus twelve hour study. Cannabinoid yields and profiles were assessed using GC 
(Appendix 1). 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



5.4.2.9 Effect of Irradiance Level on Plant and Cannablnoid Yield 

The mercury vapour (MBFU) lamps originally fitted in the glasshouse were only able to 
convert approximately 10% of the consumed electrical energy into photosynthetically 
active radiation. This compared to 30% with more modern High Pressure Sodium 
(HPS) and metal halide (MH) lamps. By replacing each 1000 watt MBF fitting with two 
600 watt high pressure sodium (HPS) or metal halide (MH) units the electricity 
consumption within the glasshouse would have been raised by 20% to the maximum 
capacity of the existing power supply. However, the increase in irradiance achieved by 
new fittings would have theoretically been three-fold, taking levels from 16 to 53 W m" 2 . 

To measure the effect of introducing such improvements to irradiance levels, identical 
batches of plants were grown in the glasshouse and in a walk-in growth chamber. The 
latter was equipped with a combination of HPS and MH lamps to deliver an irradiance 
level of 70 W m" 2 at the plant canopy. This equates to the light level typically existing in 
the glasshouse, as equipped at the time, during mid-afternoon on a bright summer day. 

The experiment commenced in September and finished in December so as to recreate 
a harvest date when the previous year's findings suggested that glasshouse yields 
would be at or near their minimum. Records from the Priva Glasshouse Control 
computer system showed that on rare very clear days, noon irradiance levels inside the 
glasshouse peaked at approximately 50 W m" 2 . However a maximum of approximately 
25 W m" 2 was more common, with morning and evening levels falling to a minimum of 
17 W m" 2 . Irradiance levels were measured using a hand-help Skye SKE 500 light 
metre at ten locations on the surface of the crop canopy. 

One hundred rooted cuttings of each of the THC chemovars G1 and the CBD 
chemovar G5 were placed in pots of growth medium following the standard procedures 
for botanical raw material production. Fifty of each chemovar were propagated in the 
growth room and the remainder were propagated in the glasshouse along-side the crop 
being routinely grown for botanical raw material production. In both the glasshouse 
and growth room the temperature was maintained at 25.0 ± 1.5°C throughout the trial 
period. Plants were placed on a bench at maximum density (pot-to-pot) for the first 
three weeks and maintained in a twenty-four-hour-per-day lighting regime. The THC 
chemovar plants were then spaced out to ten plants per square metre or seventeen per 
sq metre (G5 CBD chemovar) for the remaining eight weeks before harvest. During 
these eight weeks the lighting regime was maintained at 12 hours light/12 hours dark 
per day. Plants in both areas were watered by hand. No additional plant nutrient was 
given to the plants in either growing regime. 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



Plants were harvested after the usual eight-week flowering period and dried and 
weighed. Mixed samples of stripped foliar and floral dry material of each chemovar 
from both regimes were analysed for cannabinoid content using HPLC. 

In a subsequent larger scale test 250 m" 2 of the glasshouse was converted to raise 
supplementary lighting levels from 16 to 53 W m" 2 . Temperature and daylengths were 
kept identical in both areas. The monthly average crop yields in both areas were 
compared over the following year. 

5.4. .2. 10 Effect of the length of flowering period on the cannabinoid profile of 
heterozygous plants of the mixed THC/CBD chemotype. 

In addition to clones specifically bred to produce a mixed THC/CBD profile, one was 
discovered by chance (code name G159) while screening plants grown from 
commercially available seed. This was marketed for outdoor growing in the UK. 
Although most seedlings from this variety were found to be of the THC chemotype, five 
plants were of the BrB D genotype, as described by de Meijer et al. (2003). These 
produced plants containing THC and CBD in ratios varying between 1.5:1 and 1:1.5. 
These were adopted for detailed study in this thesis as they could be included in indoor 
and outdoor tests. Four rooted cuttings of each were grown in five litre pots of the 
standard growth media. Plants were kept in a walk-in growth room in continuous 
lighting for the first three weeks. A twelve hour light/ twelve hour dark regime was then 
implemented. Temperature was maintained at 25.0 ± 1.5 e C. Using high pressure 
sodium lamps, irradiance levels were kept at 75 W m" 2 at foliage height. After four 
weeks in short day length a program of weekly sampling commenced, one entire 
inflorescence being removed from the side of each plant. These were analysed for 
their cannabinoid content by gas chromatography (Appendix 1). 

5.4.2. 1 1 Statistical Analysis 

Analyses of variance (ANOVA), paired t-tests and F-tests were used as appropriate, 
utilising Microsoft Excel 2003 related software. 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



5.5 RESULTS AND DISCUSSION 

5.5. 1 Comparison of the Yield and Uniformity of Plants Grown from 
Cuttings or Seeds 

The yield of raw material obtained from plants grown from seed (494 g m" 2 ) and 
cuttings (515 g m" 2 ) was very similar. An analysis of variance (n = 30) showed this 
small difference not to be significant (p > 0.05). However, the mean THC content of the 
cloned plants (14.6% THC w/w) was significantly higher (ANOVA, p < 0.01) than those 
grown from seed (11.1% THC). As a consequence, the cloned plants produced 
significantly more THC per unit area - 75.4 g m" 2 (p < 0.01 , ANOVA) than those from 
seed - 54.9 g m" 2 . Clone M1 was one of five originally selected for further testing from 
approximately one hundred female plants of the variety G1. The selection was on the 
basis of its favourable ratings for vigour, yield, glandular trichome density, THC content 
and purity. The THC content of these one hundred plants had exhibited a normal 
distribution. It is not clear from this investigation whether the increased THC yield from 
the cloned material was entirely as a result of this selection process. 

Only THC, CBG and CBC were found in detectable quantities in all samples. The mean 
CBG content seed sown plants was very similar to that collected from cloned plants, 
and no significant difference was found. Although the mean CBC content of cloned 
plants was 15% higher than that of seed derived plants, this increase was not 
significant (ANOVA, p > 0.05). F-tests showed the CBC potency of seed derived plants 
to be significantly more variable (p < 0.01). The difference in variability of CBG content 
of seed sown plants was much less pronounced. Seed derived plants were not 
significantly more variable in CBG potency than those raised from clones (p > 0.05). 
When the ratios of cannabinoids in the seed-sown and cloned plants were compared it 
was found that the cannabinoid profile of seeds-sown plants was more variable than 
that those raised from cuttings. F-tests showed that the CBC:THC ratio and CBG:THC 
ratios to be significantly more variable in plants grown from seed (p < 0.01 ). 

To meet acceptable quality standards, phytomedicines have to undergo 
standardization process, this being - 

the establishment of reproducible pharmaceutical quality by comparing a product with 
established reference substances and by defining minimum amounts of one or several 

compounds or groups of compounds (or in some cases) a maximum and minimum 

amount (Heinrich etai, 2004). 



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The efficacy of cannabis can be attributed to more than one cannabinoid. Two of 
these, THC+CBD, have been shown to act together synergistically. (Williamson, 2001; 
Musty, 2004). The interaction of the other pharmacologically active cannabinoids is 
less well understood. Where active ingredients within a medicine act synergistically, 
alterations to the ratio of the synergists can have a greater effect than in a medicine 
where two active ingredients act additively. This investigation shows that the ratio of 
synergists is more strictly controlled in cloned plants. When pharmacologically active 
ingredients are extracted from botanical raw material, it could be argued that the overall 
secondary metabolite profile of the entire batch is important and that the profile of 
individual plants within that batch is of minimal significance. More work would be 
required to test this hypothesis. 

5.5.2 Effect of Irradiance Level on Plant and Cannabinoid Yield 

During the first year of regular propagation of cannabis chemovars the average 
monthly yield was recorded. A large seasonal yield variation was observed (Figure 
5.5) with winter crops yielding less than those grown in summer. 

Routine monthly analysis of cannabinoid content by GC showed that the potency of 
winter crops was as little as half that of summer crops. As a consequence of the 
combined drop in crop-yield and potency the cannabinoid yield of winter crops was 
found to be roughly a quarter of that achieved in summer (Figure 5.6). 





500 - 




450 




400 


E 


350 


57 


300 


tf> 


O) 


250 


T3 
d> 


200 


>- 


150 




100 




50 - 








1 



i 



■ CBD 

□THC 



Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 

Harvest Month 



Figure 5.5. Average monthly BRM yield (two to four crops per month) (± SD) of THC and 
CBD chemovars during the first full year of propagation. (No THC chemovar was harvested in 
April and no CBD in February and November.) 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 





70 




60 




50 


E 




0) 


40 


D) 




■a 


30 






> 


20 




10 









la 



]THC 
]CBD 



Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 

Harvest Month 



Figure 5.6. The seasonal variation in cannabinoid yield of THC and CBD chemovars during the 
first full year of propagation. Values shown were estimated by combining the average monthly 
Botanical Raw Material yield (two to four crops per month) and the average monthly THC or 
CBD content (w/w). 



Table 5.4. shows the yield and potency of identical batches of crops propagated in the 
glasshouse and in the growth-chamber. The glasshouse light level varied according to 
time of day and outside lighting conditions, with mercury vapour lamps providing up to 
17 W m" 2 . A theoretical maximum irradiance level of 50 W m" 2 was achievable in the 
glasshouse at noon on a bright winter day with supplementary lighting operating. An 
actual maximum of approximately 25 W m" 2 was more typical. Growth room conditions 
were much brighter at 70 W m" 2 . These crops were harvested during November and 
December when glasshouse grown yields were close to their minimum. 



Chemovar 


Location 


Daytime 
Irradiance 


BRM Yield 


Cannabinoid 
Potency 


Cannabinoid 
Yield 






W m" 2 PAR 


g m" 2 


% w/w 


g m" 2 


THC 


Glasshouse 


17- 50 


188 


11 


23 


THC 


Chamber 


70 


397 


16 


62 


CBD 


Glasshouse 


17-50 


157 


6 


9 


CBD 


Chamber 


70 


251 


11 


28 



Table 5.4. A comparison of Botanical Raw Material yield and potency of a THC and a CBD 
chemovar when grown in two irradiance levels during winter. 



Following this compelling observation that increased irradiance resulted in such a large 
increase in yield of plant material and cannabinoid, part of the glasshouse 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



supplementary lighting system was upgraded. Existing mercury vapour lamps were 
replaced with high pressure sodium lamps giving a greatly improved light output of a 
level similar to that achieved in the growth room test. During the following winter 
months, ten batches of each of the THC and CBD chemovar crops (> 50 plants per 
batch) were grown under both the mercury vapour lamps delivering 17 W m" 2 PAR and 
the brighter high pressure sodium supplementary lighting system delivering 55 W m" 2 
PAR. Under the mercury vapour lamps the THC and CBD chemovar yields were 234 (± 
25.6 SD) g m" 2 and 162 (± 46.6 SD) g m" 2 respectively. Under high pressure sodium 
lamps this increased to 478 (± 71 .2 SD) g m" 2 and 410 (± 82.6 SD) g m" 2 . In a paired t- 
test the improvement in yield of both chemovars under sodium lamps was highly 
significant (p< 0.01). 

The crop yields were monitored over twelve months, following the complete conversion 
of the glasshouse to the new lighting regime. The average monthly yields achieved 
before and after the lighting improvements are shown in Figure 5.7. 



700 




□ Mercury 
■ Sodium 



Harvest Month 



Figure 5.7. The average yield of the THC chemovar before and after the replacement of mercury 
vapour lamps (17 W rn 2 ) with high pressure sodium lamps (55 W m" 2 ) of improved 
supplementary lighting (± SD). The mean is typically for four crops per month. No crop was 
harvested in April of the first year. 

This showed that the monthly average yields under the brighter HPS lamps were 
significantly much greater than those previously obtained under the mercury vapour 
fittings (t-test, p < 0.001). This was even the case in the late summer months when 
abundant natural light was available. Even on the brightest days, when outside light 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



levels reached 85 klux or more, the supplementary lighting was activated when outside 
light conditions fell below 60 klux - this always being the case in the early and late part 
of the day. The amount of total light energy to which plants were exposed daily was 
thus greatly increased. 

The new lighting regime also significantly improved the uniformity of monthly average 
yields over the year (F-test, p = 0.013). However, despite this there was still some 
seasonal variation in yield. The average THC-chemotype summer yield (harvested 
May - October 573 g m" 2 ) was significantly greater (ANOVA, p < 0.001) than that of 
crops harvested over the rest of the year - 516 g m" 2 . In theory, further increases in 
supplementary lighting would have increased winter yields. Alternatively uniformity 
could be improved by reducing the irradiance levels in summer, by less use of 
supplementary lighting or increased use of glasshouse shading. 

The yields achieved in the first full year of crop growth in a solid building with no natural 
lighting were similarly monitored. Yields showed a downward trend, commensurate 
with the manufacturer's predicted age-related fall in irradiance from the lamps. A 
comparison of the variability in monthly yields of crops grown entirely indoors under 
lamps initially delivering 75 W m" 2 , with those grown in the glasshouse under HPS 
supplementary lamps, revealed no significant difference (F-test, p = 0.05) between the 
two growing environments. 

Having demonstrated such a clear correlation between irradiance levels and cannabis 
growth, the initial seasonal yield fluctuation prior to lighting improvements was 
reconsidered. With hindsight it was striking that crop yields were seen to peak in those 
crops harvested in August and September, a few weeks earlier than if grown outside at 
the same latitude. The peak in glasshouse average daily irradiance levels would exist 
around the summer solstice, five to nine weeks earlier, in late June. This suggests that 
the irradiance conditions at the very beginning of flowering have the greatest potential 
impact on the final yield. This supposition is supported when the clearly-similar 
seasonal pattern of glasshouse yields (prior to lighting improvements) is plotted 
alongside the seasonal variation of irradiance conditions existing at the 
commencement of flowering of each of these glasshouse crops (Figure 5.8). It would 
appear that Cannabis sativa has evolved to make maximum use of the light energy 
available, during the longest and brightest days of mid-summer, to develop as dense a 
foliar canopy as possible. In the following generative (flowering) phase the additional 
foliage formed would undoubtedly intercept more light energy. This would increase the 
photosynthetic ability of the plant, some of which would be diverted into secondary 
metabolite biosynthesis. The later senescence of the additional foliage would enable 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



proportionally more metabolites to be translocated via the phloem to the inflorescences 
for secondary metabolite biosynthesis. 




ance 



Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 
Month Plants Commenced Flowering 



Figure 5.8. Pattern of irradiance level in the glasshouse between 7 am and 7 pm (prior to the 
improvements in supplementary lighting) and the pattern of average monthly yields of THC 
chemovar raw material + SD (n = 4). 

This data was alternatively viewed by plotting average monthly yield as a function of 
the glasshouse light level at the beginning of flowering (Figure 5.9). On both axes the 
data was expressed as a percentage of the maximum observed. A regression 
calculation confirmed the highly significant close-to-linear correlation (R 2 = 0.96, p < 
0.001) between monthly irradiance level at the commencement of flowering and the 
subsequent final yield. 

This clear linear correlation complemented the findings of growth room studies by 
Lydon etal. (1987) who showed that cannabis assimilated carbon dioxide linearly up to 
a photon flux density of approximately 500 pmol m" 2 s~\ 400 - 700 nm. This is 
equivalent to solar radiation levels of approximately 100 W m" 2 and is well above the 
maximum daily-average irradiance encountered in this study. It is assumed that the 
linear assimilation of carbon dioxide observed by Lydon etal. (1987) corresponded to a 
linear increase in photosynthesis. A subsequent increased accumulation of both 
primary and secondary metabolites would be expected, although the relative 
proportions of these would possibly differ. The increased potency of plants grown in 
brighter conditions observed in this study supports the Carbon Nutrient Balance 
hypothesis proposed by Bryant et al. (1983). This predicts that the increased net 



124 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



photosynthesis results in an increased Carbon/Nutrient ratio within the plant. This 
favours the development of carbon-based secondary metabolites. 




Irradiance %of Max 



Figure 5.9. The average monthly yield as a function of the glasshouse light level at the 
beginning of flowering. On both axes the data was expressed as a percentage of the maximum 
observed (r 2 = 0.92, p < 0.001). 

The requirement for such high levels of supplementary lighting is understandable. At 
latitude 50 e C, ignoring effects of cloud cover, average solar radiation levels at sea level 
are approximately half those encountered at 30 e C (Albuisson et at., 2006). Although 
Cannabis sativa grown for fibre or seed is often planted at this latitude, the THC- 
chemotype grown outdoors for its cannabinoid content is more commonly found at 
latitudes of 30 e or less (Small and Beckstead, 1973a,b). In addition to unfavourable 
latitude, light transmission through a typical glasshouse roof is further reduced by at 
least 30% (Heuvelink et at., 1995). Supplementary lighting has been increasingly used 
in commercial glasshouses in the UK for food-crop production. Rarely do these deliver 
light levels more than 12 W m" 2 , but recent installations of 25 W m" 2 have been reported 
(Vale, 2008). The installation of a lighting system delivering 55 W m" 2 over an area of 
5000 m" 2 , as used here, was highly unusual and possibly unique in the UK. 

However this lighting level is less than that typically utilised by illicit growers. Evidence 
from indoor UK cannabis-growing scenes of crime (private communication) show much 
brighter lighting conditions. These appear to comply with the illumination levels 
recommended in cannabis growing guides (Green, 2003), which commonly suggest the 
use of one 600W high pressure sodium lamp per square metre of flowering crop. 



125 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



Hough et al. (2003) quotes an experienced illicit UK cannabis grower as saying that "A 
decent grower can quite easily get one gram of dried flower head per watt of lighting 
used." This refers to electrical energy consumed per unit area. It implies that the use of 
one 600 Watt lamp per square metre could result in the production of 600 g m" 2 of dry 
mature cannabis flower head. Such illicit yields are regularly claimed (Rosenthal, 
2001) and appear credible. Six hundred watt HPS lamps typically convert about 30% 
of the electricity consumed into photosynthetically active radiation (Langton and Fuller, 
2001). The irradiance in such a situation would therefore be 180 W m" 2 PAR, i.e. 
approximately twice that encountered by the pharmaceutical crop studied for this 
thesis. This highlights the typical illicit growers' desire for yield over uniformity. This 
may be partly to meet consumer demand. However, it is extremely common for illicit 
growers to be using 'extracted' (stolen) electrical energy, and as such energy 
consumption and cost is often not a consideration. 

5.5.3 Effect of Duration of Flowering Period on BRM and Cannabinoid 
Yield 

Between the sixth and eigth week of flowering the 33% extension in flowering duration 
resulted in a mean cannabinoid yield increase of over 50% (Figure 5.10). In all clones 
this extra two weeks flowering was clearly advantageous. A further 25% increase in 
flowering period from eight to ten weeks resulted in a mean increase in THC yield of 
31%, but for approximately half of the clones, including Sativex-related G1M1 and 
G2M7, the observed THC yield increase was less than 25% and the economic benefit 
of the extra two weeks flowering was doubtful. Paired t-tests showed that the mean 
increases in yield, from six to eight weeks and from eight to ten weeks, for the twenty 
five clones were highly significant (6-8 weeks p < 0.001 ,8-10 weeks p = 0.032). 



126 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



100 -i 
90 
80 
70 




Figure 5.10. The yield of THC achieved by each of the clones (n=5) after six, eight and ten 
weeks in flower. For clarity, the clone lines have been sorted in order of descending THC 
yields after ten weeks in flower. (Paired t-test 6-8 weeks p < 0.001 and 8-10 weeks p < 0.01). 



The clones that clearly benefited from a longer ten week flowering period were 
generally those reported to have an equatorial and subequatorial areas provenance. In 
their natural habitat these would experience a longer growing season. However, as 
with all cannabis varieties that are used for illicit purposes, these variety names are not 
registered in the International Code of Nomenclature for Cultivated Plants, 1995 
(Snoeijer, 2002). Thus true provenance cannot be authorised and caution is required 
when referring to the properties of such varieties. 

5.5.3. 1 Effect of Duration of Floweringt Period on Ratio of THC and CBG in THC 
Chernov ars 

Between the sixth and ten week of flowering, the mean average content of both THC 
and CBG increased in the twenty five clones. The mean proportion of CBG in the 
cannabinoid profile fell from 3.66% to 3.24% between the sixth and eighth week of 
flowering and fell further to 2.66% in the tenth week. Both decreases were highly 
significant (p = 0.0208 and p = 0.0003 respectively in two-tailed t-tests). This was 
probably due to the fact that as plant development slowed, CBG was being converted 
into THC faster by THC synthase than it was being renewed by 



127 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



geranylpyrophosphate:olivetolate geranyltransferase. The relative proportions of CBG 
and THC in a pharmaceutical product must meet a tight specification to ensure its 
quality, safety and efficacy. This investigation shows that the ratio of these two 
cannabinoids is not genetically fixed and that harvest timing can have a significant 
effect. 

Plant genetics were shown to have a greater impact on THC:CBG ratios than harvest 
timings. For example, the THC:CBG ratio of clone G44-13 was approximately 1 :200 at 
all harvest timings whereas G48-12, G48-13 and G41-14 showed a ratio greater than 
1 :20. When the THC:CBG ratios from all three harvest date were combined (Figure 
5.1 1) highly significant differences were observed between clones. 



o 

O 
+ 
O 
X 



to 
co 

O 
X 



100 
99 
98 
97 
96 
95 
94 
93 
92 
91 
90 



Sis 



SI 



E S 5 



CO 




CO 




CO 




^1" 


CD 


in 






CO 




in 


CM 


CO 


CO 




O) 


in 


CD 


CD 


CM 


CO 








in 


i 






o 




o 


C\l 












o 


d) 


CD 


C\J 


Cvj 


dj 


d) 








4 


in 






4 


CM 




4 


in 


^- 


in 




CD 




CO 


m 


^- 


^- 


in 


m 


in 




CO 


CO 




G4 


G4 


CD 


o 


G4 


CD 


CD 


G4 


CD 


O 


CD 




G4 


CD 


G4 


CD 


CD 


CD 


CD 


CD 


O 


O 


G4 


G4 


G4 


* 
















* 
















*** 


** 


** 


** 


*** 


** 


* 


** 


*** 



Clone 



Figure 5.11. A comparison of the mean relative proportions (±SD) of THC and CBG in twenty 
five clones at three harvest dates. Analyses of variance (one-way) compared the proportion of 
THC in each clone to that in the Sativex-dependent clone Gl (shown in red). (* p < 0.05, ** p 
<0.01, ***p< 0.001). 



In some cases (G44-13 v G44-16 and G50-3 v G50-4 or G50-5), there were highly 
significant differences in the THC:CBG ratios of clones derived from single varieties 
(ANOVA, p < 0.01). This supports the data reported in Section 5.5.1 which indicates 
that uniform drug feedstock is more likely to be produced by plants grown from clones 
rather than seed. The especially high degree of variability in varieties G44 and G50 
(provenance unknown) was possibly due to the parent-crosses having a higher degree 
of heterozygosity. 



128 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



5.5.3.2 Effect of the length of flowering period on profile of heterozygous chemotypes 
with mixed THC/CBD profiles 

Four weeks after being placed in twelve hour days, inflorescence development was 
rapid and capitate stalked trichomes were becoming abundant. This suggested that the 
plants were actively biosynthesising cannabinoids. Sampling commenced and was 
continued for the following six weeks. By the tenth week in short day length, no more 
viable stigmas were observed, indicating that inflorescence development and capitate 
stalked trichome formation had ceased. Table 5.5 shows that there were minimal 
increases in cannabinoid concentration in the last two weeks of the sampling period, 
but prior to that, cannabinoid contents had escalated rapidly. 

In clone G 159/1 the CBD/THC ratio remained constant over the entire flowering phase. 
However, in G 159/9 and G 159/1 6 there were significant proportional increases in CBD 
content (regression p < 0.05). Conversely, in G159/1 1 and G159/12 the proportion of 
CBD in the cannabinoid profile showed significant decreases with time (regression p < 
0.05). The stable cannabinoid profile exhibited here by clone G159/1 suggests that 
single clones of the mixed CBD/THC chemotype can be used as phytopharmaceutical 
feedstocks. However, to produce such clones the plant breeder has the combined 
challenge of identifying genetics that not only exhibit a desired cannabinoid profile but 
also deliver this reliably and uniformly during the growing process. In the absence of 
such clones, desirable cannabinoid mixtures can be only be achieved by blending 
materials with differing profiles. 



129 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



Weeks 
in 12 h 
days 


Mean 
Cannabinoid 
Content % 
w/w (± SE) 


Clone 


G159-1 


G159-9 


G1 59-11 


G159-12 


G159-16 


CBD as % of CBD+THC (± SD) 


4 


1.55 
(± 0.08) 


61.78 
(±0.14) 


58.79 
(± 0.70) 


61.41 

(±0.37) 


42.60 
(±0.35) 


59.49 
(± 0.49) 


5 


3.12 
(± 0.69) 


61.43 
(±0.41) 


58.55 
(±0.29) 


62.21 
(±1.14) 


43.14 
(± 0.63) 


60.02 
(±0.80) 


6 


5.69 
(±1.27) 


62.03 
(± 0.85) 


59.65 
(±0.15) 


61.20 
(± 0.22) 


42.95 
(± 0.49) 


61.19 
(±0.54) 


7 


9.22 
(±1.22) 


61.84 
(± 0.38) 


62.05 
(± 0.38) 


59.23 
(± 0.30) 


43.06 
(± 0.44) 


61.49 
(±0.11) 


8 


11.15 
(±1.08) 


61.53 
(± 0.09) 


62.79 
(±0.21) 


58.91 
(± 0.57) 


41.86 
(± 0.90) 


62.21 
(±0.58) 


9 


11.65 
(± 0.92) 


61.32 
(± 0.37) 


63.00 
(± 0.95) 


59.11 
(±0.58) 


42.06 
(± 0.05) 


62.17 
(± 0.23) 


10 


1 1 .95 
(± 1 .24) 


61.99 
(±0.53) 


64.16 
(± 0.73) 


59.60 
(±1.22) 


41.08 
(±1.17) 


62.29 
(± 0.20) 




Regression 


No 
significant 
change 

p> 0.05 

r = 0.03 


Significant 
Increase 

p< 0.01 
r = 0.96 


Significant 
Decrease 

p< 0.05 
r = 0.81 


Significant 
Decrease 

p< 0.05 
r = 0.79 


Significant 
Increase 

p< 0.05 
r = 0.95 



Table 5.5. The relative proportions of CBD and THC during plant development in five clones 
derived from variety G159. Results are shown as the proportion of CBD expressed as % of 
CBD+THC (± SD). The regression calculations test the significance of the changing proportion 
of CBD and THC in each clone, between the 4 th and 10 th week in 12 h daylength. 



5.5.4 Effect of Daylength on Plant Development and Cannabinoid Profile 

5.5.4. 1 Comparison of Twelve and Thirteen Hour Daylength Regimes 

Despite their varying provenance, all the varieties included in this investigation 
commenced to flower within ten days of being placed in a thirteen-hour day length. 
This suggested that all had a 'critical daylength' of over thirteen hours. As shown in 
Table 5.6 a daylength of thirteen hours occurs naturally at around August 18 th , at a 
latitude of 26 e N, and progressively later at more northerly locations. Had a Columbian 
variety been included, perhaps originating for Santa Marta (latitude 1 1 ° north), it is 



130 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



possible that it would not have flowered - as even at the summer solstice the day 
length is just 12 hour 45 minutes. The critical day length for plants from this latitude 
would be therefore expected to be substantially less than thirteen hours. It is possible 
that the plant would have eventually have flowered in response to plant age rather than 
daylength, as is common for tropical varieties. 

All of the locations cited in Table 5.6 have been important in the breeding history of the 
varieties currently developed for pharmaceutical use. Skunk #1 and subsequently 
some of the other varieties within this test were originally bred using a mixture of 
Columbian, Mexican and Afghan genetics (de Meijer, 1999; Clarke, 2001). Despite the 
Columbian inclusion, all varieties appear to have inherited from their more northerly 
ancestors an ability to flower readily in a thirteen-hour daylength. The CBD-rich variety 
G5 was bred from landrace plants derived from the Black Sea coast in Northern 
Turkey, where thirteen hours day length occurs in early September, a few days earlier 
than in Southern England. 



Latitude: - 


11 e 


26 s 


36 e 


41 s 


51 e 


Climate: - 


Tropical 


Semi-tropical 


Temperate 


Location: - 


Santa Marta, 
Columbia 


Monterrey, 
Mexico 


Mazar al Sharif, 
San Fransisco and 
Ketama* 


Samsun, 
Turkey 


London, 
UK 


Jul-15 


12.43 


13.35 


14.25 


14.50 


16.10 


Aug-01 


12.35 


13.19 


14.05 


14.20 


15.24 


Aug-15 


12.28 


13.03 


13.37 


13.49 


14.37 


Sep-01 


12.19 


12.38 


12.59 


13.06 


13.34 


Sep-15 


12.11 


12.18 


12.27 


12.28 


12.40 


Oct-01 


12.01 


11.54 


11.48 


11.45 


11.37 


Oct- 15 


11.53 


11.34 


11.19 


11.10 


10.45 



Table 5.6. The falling late-summer daylength (hours.minutes) in a range northern hemisphere 
cannabis growing areas. (* Contrasting locations at similar latitude in Afghanistan, USA and 
Morocco.) 

Although floral development of all plants commenced equally promptly in a twelve or 
thirteen-hour daylength, the pattern of floral development differed greatly in the two 



131 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



regimes. The visual assessments the degree of stigma senescence (Table 5.7) 
showed that the rate of flower formation slowed more rapidly in the twelve-hour regime. 
Assessed eight and ten weeks after being placed in short daylength, the mean 
proportion of senescence stigmas was significantly higher in the twelve hour daylength 
(paired t-test, p < 0.01 , at both assessment dates). The difference was most dramatic 
in clone line M84, which after ten weeks in a twelve-hour daylength, had completely 
ceased to develop new flowers whereas the same clone in thirteen hours daylength 
continued to flower prolif ically. 





Eight weeks in Short Days 


Tenth Week in Short Days 




12 hr Day 
length 


1 3 hr Day 
length 


1 2 hr Day 
length 


1 3 hr Day 
length 


Clone 










M1 


60 


33 


50 


45 


M6 


60 


38 


80 


45 


M57 


80 


35 


80 


55 


M59 


70 


50 


35 


10 


M60 


95 


35 


100 


98 


M61 


99 


38 


100 


75 


M79 


70 


15 


60 


50 


M82 


20 


4 


30 


15 


M84 


50 


3 


100 


10 


M87 


30 


4 


25 


5 












Mean 


63.4 ** 


25.5 ** 


66 ** 


40.8 ** 



Table 5.7. A comparison of the proportion of senesced stigmas observed on ten clone when 
induced to flower in daylengths of twelve or thirteen hours. Assessments were made eight and 
ten weeks after the plants were placed in short daylength. ** Significant difference (p < 0.01, 
paired t-test). 

A twelve-hour day length occurs at all northern hemisphere latitudes in the last week of 
September (Table 5.6), at around which time a cannabis crop grown for seed would 
typically be harvested (Bocsa and Karus, 1998). Any female flowers formed after early 
September would be unlikely to have sufficient light and warmth to produce viable seed 
before the plant died. Moreover, pollen formation typically ceases before the end of 



132 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



August after which time male plants typically die thereby not competing with the female 
plants as they set seed. The formation of further female flowers would be a futile waste 
of plant resources. 

After eight weeks in short daylength there was no significant difference in the mean 
height of the ten clones in twelve or thirteen hour days (paired t-test). After ten weeks 
significant differences were observed in the height of five clones (ANOVA), and this 
was most pronounced in vigorous F1 hybrids M82 and M87 (Figure 5.11). As a 
consequence there was a significant difference in the mean height for all ten clones in 
the two regimes (paired t-test p < 0.05). M82 and M87 were much taller than other 
clones. Excessive height is a disadvantage in a glasshouse grown crop and the longer 
daylength exacerbated this problem. Tall plants are difficult to support and to handle, 
and are less easily examined for pest and disease. 




Figure 5.12. Effect of Daylength on Plant Height ± SD (n = 20) ten weeks after induction of 
flowering (* p < 0.05, *** p < 0.001, ANOVA for individual clones and paired t-test for the 
overall mean). 



Eight weeks after flower initiation, minimal differences were observed between the 
botanical raw material yields of plants grown in twelve and thirteen hour daylengths. 
The two F1 hybrid clones M82 and M87, that had shown large differences in height, 
showed marked increases in yields at the longer daylength when harvested ten weeks 
after flower initiation (Figure 5.13). However, overall the longer daylength did not 
significantly increase the mean yield of the ten clones (paired t-test, p > 0.05). 



133 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



rr 



1400 
1200 
1000 
800 
600 
400 
200 




J 



CD 




0) 


o 




0) 


C\J 










LO 


LO 


CD 


CD 




00 


00 


00 


























* 



















■ 12 hours 

■ 13 hours 



03 
CD 



Figure 5.13. Effect of Daylength on Yield of Botanical Raw Material ± SD (n = 5 plants) ten 
weeks after induction of flowering (* p < 0.05, *** p < 0.001, ANOVA). 



The cannabinoid content of the higher yielding clones M82 and M87 was greatly 
reduced in the longer daylength and this resulted in a marked decrease in mean 
cannabinoid yield of the ten clones at both harvest dates (Table 5.8). Conversely the 
clone lines M1 and M6, showed a possible yield benefit. When assessing the overall 
effect of daylength on the mean cannabinoid yield of the ten clones, the paired t-tests 
showed no significant advantage for either regime. 



134 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



Clone 


8 weeks in flower 


10 weeks in flower 




1 2 hr days 


1 3 hr days 


12 hr days 


13 hr days 


M1 


57 


79 


70 


73 


M6 


44 


54 


65 


83 


M57 


64 


64 


51 


87 


M59 


52 


34 


54 


30 


M60 


41 


45 


36 


39 


M61 


29 


30 


32 


31 


M79 


50 


53 


43 


49 


M82 


84 


33 


122 


63 


M84 


34 


35 


55 


53 


M87 


84 


38 


100 


73 


Mean 


54 


47 


62 


58 



Table 5.8. Effect of Daylength on cannabinoid yield (g m" 2 ), eight and ten weeks after induction 
of flowering. 

Daylength had highly significant effects on cannabinoid profile. Harvested eight or ten 
weeks after being placed in a twelve or thirteen hour daylength, all ten clones had a 
proportionally higher CBG content if grown in the thirteen hour regime (Table 5.9). The 
mean difference was significantly higher after eight weeks (p < 0.01) and ten weeks (p 
< 0.05, paired two-tailed t-tests).This was likely due to the fact that more new florets 
and accompanying glandular trichomes were being formed in the thirteen hour 
daylength, and these would be synthesising more CBG. Conversely plants in the 
thirteen hour regime possessed proportionally less THCV (p < 0.001 after eight weeks 
and p < 0.01 after ten weeks). This finding supports other results (not reported here) 
that propyl cannabinoid synthesis is more rapid than that of pentyl cannabinoids. As a 
consequence, in aging tissue the biosynthesis of THCV reaches completion before that 
of THC. 



135 



Chapter 5. Indoor Propagation of Medicinal Cannabis 





CBG as % of CBG + THC 




THCV as % of THCV + THC 


Daylength 


12 h 


13 h 


12 h 


13 h 




12 h 


13 h 


12 h 


13 h 


Time in Short Days 


8 weeks 


10 weeks 




8 weeks 


10 weeks 


M1 


1.00 


1.20 


0.65 


2.00 




0.52 


0.48 


0.55 


0.46 


M6 


0.77 


1.40 


0.81 


1.25 




0.65 


0.57 


0.73 


0.53 


M54 


3.96 


4.38 


3.30 


3.97 




0.39 


0.34 


0.33 


0.29 


M57 


0.69 


0.98 


0.45 


0.82 




0.42 


0.34 


0.51 


0.35 


M59 


1.27 


2.63 


1.11 


0.96 




0.47 


0.45 


0.57 


0.42 


M60 


3.30 


4.57 


2.51 


2.59 




0.54 


0.52 


0.54 


0.48 


M61 


1.53 


3.43 


0.98 


1.44 




0.29 


0.20 


0.32 


0.18 


M79 


1.55 


3.78 


1.29 


1.57 




0.43 


0.37 


0.45 


0.37 


M82 


5.77 


7.15 


4.41 


6.11 




0.32 


0.16 


0.31 


0.17 


M87 


4.10 


6.55 


3.29 


3.40 




0.39 


0.35 


0.32 


0.37 


Mean 


2.39 


3.61 


1.88 


2.41 




0.44 


0.38 


0.46 


0.36 


p, t-test 
(2 tailed) 


0.0011 


0.0176 




0.0006 


0.0015 



Table 5.9. The effect of day length during flowering on cannabinoid profile. Results shown are 
the proportion of CBG and THCV, expressed as a % of the CBG+THC or THCV+THC total, in 
ten clones after eight and ten weeks in short daylength. 

5.5.4.2 Comparison of Eleven and Twelve Hour Daylength Regimes 

Whereas reducing the daylength from thirteen to twelve hours had a dramatic effect on 
floral development, a further reduction to eleven hours only marginally accelerated the 
cessation of new flower formation, Table 5.10. 



136 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



Clone 


Eight weeks in Short Days 


Tenth Week in Short Days 




11 hr 


12 hr 


11 hr 


12 hr 


M1 


30 


30 


60 


55 


M6 


35 


40 


60 


60 


M11 


80 


70 


100 


98 


M16 


50 


38 


100 


98 


M57 


15 


8 


65 


55 


M58 


28 


30 


45 


55 


M59 


30 


40 


85 


70 


M60 


95 


80 


99 


100 


M61 


20 


25 


98 


70 


M82 


8 


10 


23 


20 


M84 


15 


10 


50 


45 


M87 


10 


12 


40 


30 


Mean 


35 


33 


69 


63 



Table 5.10. A comparison of the proportion of senesced stigmas on ten clones in twelve and 
eleven hour daylength regimes when assessed eight and ten weeks after the induction of 
flowering. Just one overall visual assessment was made for each clone. 

F1 hybrids M82 and M87 showed very large differences in height when grown in 
contrasting twelve and thirteen hour regimes. However, along with all other clones, 
these showed no significant difference in height when grown for eight or ten weeks 
(Figure 5.14) in eleven and twelve hour daylengths (ANOVA). Similarly, a paired t-test 
revealed no significant difference in the mean heights of the ten clones in either 
daylength. 

The extra hour of day length resulted in significantly greater yields of most clones 
(ANOVA) at both assessment dates. For simplicity, just the result of the latter 
assessment is shown in Figure 5.15 (* p < 0.05 and *** p < 0.001). A paired t-test 
showed the mean weight of the twelve clones to be greater in the longer day length at 
both assessment dates. 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 




□ 11 hour 

□ 12 hour 



Clone 



Figure 5.14. Effect of Daylength on Plant Height ± SD (n=20) ten weeks after induction of 
flowering. In a paired t-test there was no significant difference in the mean height of plants 
grown in 1 1 and 12 hour days. 




Figure 5.15. Effect of Daylength on Yield of Botanical Raw Material ± SD (n = 20 plants) ten 
weeks after induction of flowering. In the Analyses of Variance, the levels of significance were 
shown as * p < 0.05 and *** p < 0.001. In a paired t-test, the mean height of plants in the 12 
hours days was significantly higher p < 0.001. 



When harvested after ten weeks in the flowering regime, all clones showed a lower 
cannabinoid yield in the shorter daylength (Figure 5.16). The overall mean 22% 
reduction in cannabinoid yield was highly significant, (p < 0.01, two-tailed t-test). This 
reduction in yield could not be attributed to a prominent visible change in plant 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



morphology. Plants in the shorter eleven-hour daylength had received 9% less light 
energy per day. Conversely, these plants spent 9% longer in darkness and would be 
expected to have lost yet more energy as a result of a longer period of respiration. 
This would seem a major contributing cause of the yield reduction in the eleven hour 
daylength and is in marked contrast to the lack of yield difference observed when 
comparing the twelve and thirteen hour daylengths. In the thirteen hour regime the 
potential yield benefits of increased energy were not utilised by the plant. 




11 Hour 

12 hour 





CD 




00 




CD 


o 




CM 












LO 


m 


LO 




CD 


CD 


00 


00 


00 






i 























03 
CD 



Clone 



Figure 5.16. Effect of Daylength on cannabinoid yield ten weeks after induction of flowering 
(Paired t-test, two tail ** p < 0.01). 



The HPLC analysis method that was used to analyse the cannabinoid profile showed 
limited sensitivity to CBG and THCV, and these proved to be below the detectable level 
in some clones. Nevertheless, sufficient data was available to assess the proportion of 
CBG, as a percentage of the CBG+THC+CBD total, in six clones (Table 5.11). In a 
paired test, there was no significant difference in the mean proportion of CBG present. 



139 



Chapter 5. Indoor Propagation of Medicinal Cannabis 



Daylength 


CBG as % of Cannabinoid Total 


11 h 


12 h 


M57 


0.45 


0.90 


M58 


4.23 


4.52 


M59 


1.31 


1.33 


M60 


2.56 


2.29 


M84 


1.73 


1.67 


M87 


3.13 


2.82 


Mean 


2.24 


2.26 


Paired t-test 


p = 0.98 



Table 5.11 The effect of day length during flowering on cannabinoid profile. Results shown are 
the proportion of CBG expressed as a % of the CBG+THC+CBD total, in six clones after eight 
weeks in short daylength. 

5.5.4.3 Review of the comparisons of plants induced to flower on daylengths 11, 12 
and 13 hours. 

Amongst the major findings, from the studies comparing the effect of daylength, it was 
found that increasing the daylength from twelve to thirteen hours increased energy 
consumption by at least 8%, but resulted in no beneficial increase in botanical raw 
material yield. There was sometimes also an unwelcome increase on plant height. 
Effects of increased day length on cannabinoid yield were variable, with some clones 
showing a large decrease in cannabinoid production at the longer day length. 
Conversely, reducing the day length to eleven hours saved energy by approximately 
8% but resulted in an economically unacceptable and statistically significant 15 - 20% 
mean reduction in plant yield (p < 0.01). There was also a significant reduction in the 
mean cannabinoid yield (p < 0.01 after 10 wks in short days). 

Placing indoor-grown cannabis in a short daylength to induce the plants to flower is 
widely practised. The concept of flower initiation in response the arrival of the critical 
daylength, and the part that phytochromes play in this are widely reported (Halliday and 
Fankhauser, 2003). The effect of short daylength on inflorescence development is less 
well studied. In addition to a critical daylength, at which point flowering is induced, this 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



study suggests that there is a previously unreported second critical daylength at which 
development of further female flowers is inhibited. 

In a glasshouse setting, where crops in various stages of floral development grow side- 
by-side, it is not practicable to recreate the steadily shortening day length experienced 
by outdoor grown cannabis. A standardised daylength has to be maintained once 
flowering has commenced. The flowering daylength adopted can dramatically affect the 
appearance of the inflorescence, with plants in thirteen hours day length continuing to 
produce significantly more fertile female flowers for much longer than those in twelve or 
eleven hour regimes (p < 0.05). An assessment of the proportion of senesced stigmas 
in an inflorescence is often regarded as useful when deciding upon harvest date of illicit 
crops. However, this investigation concluded that stigma senescence is not useful for 
judging the cannabinoid content of crops grown for pharmaceutical purposes. 

Eleven or thirteen hour daylength regimes are less economical in producing cannabis 
for the production of cannabinoid-based medicines than the twelve hour regime initially 
adopted. Future research to explore small adjustments to the adopted twelve-hour 
regime may be beneficial. Research with ornamental pot-grown chrysanthemums 
(short day plants) has shown that adjustments of just ten minutes to the daylength can 
have a dramatic effect on the economic value of the plants produced (Langton and 
Fuller, 2001). 

Rather than simply altering the lighting regime to save costs, much greater savings 
could be made by growing the crop outdoors. Whereas the glasshouse environment 
provided uniform temperature, irradiance levels and daylength the outdoor environment 
exposes the crop to variable conditions. If these variabilities were only found to affect 
secondary metabolite yield this would be tolerable. The production of plants with a 
significantly different secondary metabolite profile or poor quality would raise serious 
concerns. The next chapter evaluates this possibility. 

5.6 CONCLUSIONS 

This chapter examined the development, and more especially its secondary metabolite 
content, when grown indoors. The overall aim was to determine ways of identifying 
growing methods and harvest timings that improved yield and crop uniformity when 
growing medicinal cannabis crops indoors in the UK. 

Specific tests with the Sativex® dependent variety G1 showed that cannabinoid profile 
uniformity was significantly improved by growing plants from clones (cuttings) rather 
than seeds. 



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Chapter 5. Indoor Propagation of Medicinal Cannabis 



The duration of the flowering period prior to harvest was seen to have a statistically 
significant effect on cannabinoid profile when tested on a wide population of genotypes. 
However, limited data suggested that the proportion of CBG in the cannabinoid profile 
of Sativex-dependent varieties G1 and G2 had stabilized after eight weeks in flower. 
The ratio of THC and CBD in heterozygous plants did not remain stable during floral 
development, and this instability is one supportive reason for producing THC and CBD 
for Sativex® from separate homozygous chemotypes. 

When inducing and maintaining floral growth, a twelve hour daylength was shown to be 
more energy efficient than eleven and thirteen hour regimes. 

The most pronounced findings, from the research for this chapter, resulted from studies 
with glasshouse supplementary lighting. Significant improvements were achieved in 
the year round uniformity and yield of botanical raw material. Cannabinoid uniformity 
and yield were similarly improved. It was shown that the irradiance levels at the 
commencement of flowering had the greatest effect on yield. However, these 
improvements were only achieved by the consumption of lighting energy levels well 
above those typically used in the UK horticultural industry. One way of avoiding this 
energy use would be to grow the crop outdoors. Rather than simply altering the lighting 
regime to save costs, much greater savings could be made by growing the crop 
outdoors. Whereas the glasshouse environment provided uniform temperature, 
irradiance levels and daylength the outdoor environment exposes the crop to variable 
conditions. If these variabilities were only found to affect secondary metabolite yield 
this would be tolerable. The production of plants with a significantly different secondary 
metabolite profile or poor quality would raise serious concerns. The next chapter 
evaluates this possibility. 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



Chapter 6 . The Outdoor Propagation of 
Phytopharmaceutical Cannabis 

6.1 INTRODUCTION 

The concept of growing phytopharmaceutical cannabis outdoors was initially rejected 
due to plant quality and security concerns. However, prior to this thesis, an 
experimental outdoor trial was planted. This demonstrated that potentially high yielding 
cannabis crops could be grown in southern England (Potter, 2004), the Sativex- 
dependent clone G5 M1 1 producing 500 g m" 2 of dry BRM. This was approximately the 
same yield as that achieved in a glasshouse crop. The trial did not aim to explore the 
maximum yield that could be achieved and with improved knowledge higher yields may 
have been obtained. No attempt was made to analyse the effect of outdoor growing on 
secondary metabolite profile. In the following two seasons further batches of the clone 
G5 M11 were planted and crop establishment was good. However, both crops 
encountered fungal spoilage by Botrytis in the last week before planned harvest. 
Subsequent attempts to grow clones of the Sativex-dependent THC chemovar G1 
failed, as the plants commenced flowering far too late in the season to produce a 
worthwhile crop. However, another high-THC genotype did achieve high yields and 
produced a potential phytopharmaceutical feedstock containing 11.8 ± 1.5% (w/w) 
THC. This was close to the average potency of illicit sinsemilla cannabis circulating in 
England in 2005 (Potter et at., 2008), most of which was thought to have been secretly 
grown indoors. The first field trial also compared the establishment of plants grown 
from rooted cuttings and from seed. Both methods proved satisfactory but 
establishment from seeds was markedly less labour intensive. 

From an agricultural perspective it therefore appeared that outdoor growing of cannabis 
was feasible in England, although there was little data to demonstrate the potential 
quality of this material. Concerns remained about the security of outdoor cannabis 
crops as the medicinal cannabis crop has a potentially high cash value, and an unusual 
cachet that appeals to the curious and mischievous. Hemp-fibre and seed crops of 
Cannabis sativa grown in the UK have frequently been damaged by the public, who 
have mistaken the crop for recreational cannabis. An all-female CBD chemovar crop 
bears a closer similarity to recreational cannabis, both in appearance and odour, and 
would be more likely to be tampered with. Any pharmaceutical crop is also possibly 



143 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



vulnerable to the attention of those protesting against the animal testing associated 
with the industry. An outdoor crop would require a very discrete and secure location. 

In marked contrast to the field environment the glasshouse conditions were well 
controlled, and harvest timings could be routinely anticipated and labour timetabled 
accordingly. If the timing of harvest of field-grown crops could be predicted with similar 
confidence there would be clear benefits. When commencing this research it was not 
known how predictable the date of outdoor harvest would be, and how long crops could 
be regarded as being at the appropriate stage. As only one crop had been successfully 
harvested prior to this thesis, the predictability of harvest date was still little understood. 

It was anticipated that a major difficulty of outdoor growing would be the requirement to 
dry large quantities of material at the exact point when crops were deemed ready for 
harvest. An early attempt to dry bulk quantities of outdoor grown crop resulted in 
failure. The crops were dried using the same method employed for the glasshouse 
crop, and much of the material was lost due to fungal spoilage. Exceeding the microbial 
count threshold, as stated in the European Pharmacopoeia, is the most common cause 
of rejection of plant material (Baier and Bonne, 1996). Prior to this thesis a crop was 
successfully dried in the oast house, using a diesel fuelled oast furnace, in a similar 
fashion to that traditionally used for hops. The lower humidity and rapid drying 
conditions prevented fungal proliferation. This type of drying appeared to warrant 
further evaluation. However, although the diesel used here is an acceptable fuel source 
for the drying of food-grade hops, this would not be acceptable for a medicinal crop. 
GAP Guideline 6.6 dictated that medicinal herb crops had to be dried naturally or using 
methane, propane or butane (EMEA, 2006). For future drying experiments, alternative 
GAP-compliant methods would be necessary. 

A major advantage of growing outdoors is that it avoided the large energy cost incurred 
in the glasshouse (> 500 kW hr per kilo of dry botanical raw material) and consequently 
created a much smaller carbon footprint. At the start of this study it appeared that 
outdoor growing was feasible and, if disease and crop drying difficulties could be 
overcome, there were clear cost and environmental advantages. However, the effect of 
outdoor growing on cannabinoid and terpene profile remained untested. 

6.2 AIM and OBJECTIVES 

Trials were performed annually over four consecutive years. The overall aim was to 
enable the growth and harvest of good quality plants, with the same cannabinoid and 
terpene profile as glasshouse crops. This necessitated an improvement in the 
understanding of how outdoor growing affected plant development, secondary 



144 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



metabolite production and susceptibility to pests and disease. Specific tests would 
then be needed with the objective of overcoming these problems and improving 
harvest and drying techniques. These objectives are described in specific details as 
follows. 

6.2.1 The effect of growing environment on female plant development 

Unlike the glasshouse crop, which routinely took eleven weeks to grow from a rooted 
cutting to fully mature plant, the outdoor grown plant took up to twice as long. As such, 
specific observations were performed to ascertain how the development of outdoor 
grown cloned all-female plants compared to that of a glasshouse crop and how 
optimum time for harvest could be visually judged. 

6.2.2 Comparison of the secondary metabolite yield and profile of fresh 
plant material and enriched trichome preparations made from them 

As shown in Chapter 4, enriched trichome preparations could be produced from 
glasshouse grown crops which captured most of the cannabinoid content of the raw 
plant material. These were less bulky materials from which essential oil samples could 
be steam-distilled to allow detailed terpene analysis. Trials were performed to compare 
the secondary metabolite profile of freshly harvested plant material and enriched 
trichome preparations. If the results indicated that glandular trichomes collected from 
fresh material had the same secondary metabolite profile as the intact plant material, 
this collection method might prove more efficient than harvesting, drying and then 
processing whole plants. 

6.2.3 The effects of harvest timing on secondary metabolite yield and 
profile 

One detailed trial aimed to assess how the cannabinoid and terpene yield and profile 
changed during the latter phases of inflorescence maturation. This would help 
ascertain the growth stage at which plants were most appropriately harvested to meet 
the preset specification. 

6.2.4 Comparison of the secondary metabolite profiles of glasshouse and 
outdoor grown plants 

In 2006 studies were performed to compare the terpenoid content and profile of 
glasshouse and field grown plants, during inflorescence maturation. The objective was 
to ascertain if and how secondary metabolite content was altered by growing 
conditions. 



145 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



6.2.5 Evaluation of outdoor pest and disease issues 

The trial program would aim to find out what major pest and disease problems might 
arise and assess their potential effect on plant quality and yield. 

6.2.6 Evaluation of Crop Drying Methods 

A range of drying methods was evaluated with two objectives. The first was to find a 
way of speeding the processing of a single seasonal harvest. The second was to 
assess how the drying method might be improved, to reduce fungal spoilage. 

6.3 MATERIALS 



Materials 


Source 


Seed for cannabis varieties G1 , G5 


HortaPharm BV Schimkelhavemkade 1075 
VS Amsterdam Netherlands 


Seed for cannabis variety G159 


Undisclosed commercial source. 


Propane gas heater and accompanying 
bottled gas. 


Local hire. Location withheld for security 
reasons. 


Diplex portable handheld thermometer/ 
hygrometer (calibrated in-house) 


Diplex Ltd., PO Box 172, Watford 
England, WD17 1BX 


Carbolyte drying oven 


Carbolite - Parsons Lane, Hope, Hope 
Valley. S33 6RB UK 



Table 6.1 Propagation Materials and Equipment used in the field trials program to evaluate the 
outdoor propagation of Cannabis sativa L. 

The sieves, microscopes and analytical equipment were the same as those described 
in Chapters Two to Four. 

6.4 GENERAL AGRONOMIC METHODS 

6.4.1 Seedbed Location, Preparation and Crop Establishment 

Being mindful of security concerns, and to satisfy Home Office license conditions, the 
crop was grown in a discrete high-walled garden. Seedbeds were prepared manually 
using a fork and rake and metaldehyde-based molluscide pellets applied to the soil to 
provide slug control. 

Cuttings were raised as described in Chapter Five. These were placed outside the 
glasshouse for seven days to acclimatise to outdoor conditions. They were then 
planted in rows one metre apart at a density of four to six cuttings per square metre, as 



146 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



found adequate in first year's trial. Predictably, variable weather and soil moisture 
conditions were encountered in the first weeks of growth and plants were watered by 
hand when insufficient soil moisture was naturally available. 

6.4.2 Field Trial Design 

In the first three years single 100 m" 2 plots of clone M16 were established, and specific 
tests performed on this single clone. The 2006 trial compared the performance of the 
G5 CBD chemovar at five harvest timings. Seven plots of each regime were laid in a 
randomised block. A small area outside of the main trial was dedicated to some 
heterozygous cultivars producing approximately equal quantities of THC and CBD. All 
were derived from the single variety G159. 

6.4.3 Soil Nutrition 

All trials were given a light dressing of approximately 40kg/ha of fertilizer, containing 
34.5% w/w nitrogen, before or soon after planting. However, no account was taken of 
residual fertilizer already existing in the soil. In all years lush growth was observed and 
no deficiency symptoms were observed. 

6.4.4 Pest and Disease Monitoring and Management 

In each season the trials were examined at least once per week throughout the 
season. Pests and disease were identified and their severity noted. At no stage were 
any chemical treatments applied. Rabbits caused extensive damage to a crop planted 
in 2001 and subsequent trials were surrounded with protective fencing. Weeds were a 
constant problem and were manually removed on a regular basis each year. 

6.4.5 Harvest 

In all cases plants were cut at the base below the lowest side-branch upon which any 
floral material was observed. Plants were placed on clean sheeting prior to relocation 
to an oast house for drying. 

6.4.6 Crop Drying and Stripping 

Crops were hung to dry on suspended wires in an oast house drying chamber. 
Propane gas burners provided hot air and fans ensured that this moved freely within 
the drying environment. Excess heat was allowed to escape through controlled 
ventilation ducts in the ceiling above the drying chamber. Thermostats within the drying 
chamber maintained drying conditions at the selected temperature, and this was 
monitored (in compliance with GAP Guideline 4.6) using the oast house environmental 
management recording system. Humidity was read with a hand-held meter. 



147 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



In each of the three years 2003-2005, separate batches were dried in temperatures of 
30°, 40° and 50°C (± 2°C). At the end of the drying period the relative humidity in these 
respective regimes was recorded as being approximately 30%, 20% and 10% 
respectively. Ten samples (approximately 1 g each) were taken at three-hourly 
intervals during the drying process. Their moisture content of these was determined by 
weighing the moisture loss when dried for 24 hours at 105°C. Once judged by touch to 
be sufficiently dry, the leaf and floral material in the oast was stripped from the stems 
by hand. This material was relocated to a dehumidified store at 30-35% RH to 
equilibrate. It was expected that in this environment the samples would settle to a 
uniform predicted moisture content of 15 ± 2%, based on experience with glasshouse- 
grown crops. 

6.4.7 Assessment of Crop Development 

The 2005 field trial was visited weekly and the height of thirty randomly selected plants 
was measured. A typical plant would produce a dominant main inflorescence, and 
many shorter side branches. Plant height was taken as the height of the top of this 
central inflorescence as measured from the soil surface. In the following year's trial, 
subjective weekly assessments were made of the state of development of the 
inflorescence, from the appearance of the first stigmas through to complete stigma 
senescence. An assessment was also made of the overall proportion of senesced 
stigmas in each of the seven replicates. To compare the pattern of development of the 
field and glasshouse-grown plants, the same observations were made on the 
inflorescences of three consecutive crops of the same clone. 

6.5 Secondary Metabolite Purification and Analytical Methods 

6.5. 1 Production and Collection of Enriched Trichome Preparations 

Ten litres of crushed ice were placed in a fifty litre bucket and twenty litres of tap water 
added. Seven plants were collected at random from the field trial. Approximately ten 
litres of foliar and floral tissue were stripped from the plants. These were promptly 
plunged into the iced water and the temperature allowed to stabilize at below 1°C. The 
mixture was agitated with a food mixer at maximum speed for ten minutes. The 
resultant slurry was then poured through 220 urn and 25 urn 'Bubblebag' sieves. The 
material settling on the 25pm sieve was placed in a 100ml Duran bottle and moved to a 
refrigerator at 4°C. The procedure was repeated until all the material from the seven 
plants had been processed. A subsample was examined, using a Brunei MX3 binocular 
microscope, to check the efficiency of the process. Over 90% of the solid material 
present was judged to be glandular trichome resin heads, the remaining proportion 



148 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



consisting of capitate trichome stalks, cystolythic trichomes and fragments of mesophyl 
tissue. The ETP was then relocated to a deepfreeze at -20°C prior to analysis. 

6.5.2 Steam distillation of trichome rich preparations 

Samples were distilled at Botanix Limited, Hop Pocket Lane, Paddock Wood, Kent, UK 
according to the company's Standard Operating Procedure for the Determination of 
Volatile Hop Oil (Institute of Brewing Method). In compliance with this method samples 
were sufficiently thawed within their sealed Duran 200ml bottles to enable the contents 
to be decanted into individual one-litre round bottom flasks with a B55 neck. A few anti- 
bumping granules were added to the flasks before connecting each one to a British 
Pharmacopoeia still, using a B55/34 adaptor. The flasks were then heated using a 
heating mantle, and the contents distilled for three hours. Using this method Howard 
(1970) showed that the extraction of essential oils from hop lupulin (glandular 
trichomes) was reliably complete in this time. During this period the flow of condensate 
was controlled to cause minimum disturbance to the oil in the trap. At the end of three 
hours the volume of oil was recorded and then decanted for analysis by GC 
(Appendix 2). 

6.5.3 Steam distillation fresh foliar and floral material 

Two kilograms of each sample of fresh material and five litres of water were placed in 
ten litre round bottomed flasks and a few anti-bumping added. The flasks were then 
connected to the still using a B55/34 adaptor. The mixture was boiled for two hours, 
after which time no further volatiles were seen to be condensing within the stills. 
During this period the flow of condensate was controlled to cause minimum disturbance 
to the oil in the trap. At the end of two hours the volume of oil was recorded and 
decanted. These oil fractions were analysed for qualitative terpene content by GC at 
Botanix Ltd. The quantitative content of a small range of terpenes within this mixture 
was determined by GC at GW Pharmaceuticals Ltd (Appendix 2). 

6.6 Statistical methods 

Standard deviation, analyses of variance, and regression calculations were performed 
using Microsoft Excel 2003 software. 

6.7 RESULTS and DISCUSSION 

6.7.1 Observations on Crop Establishment and Plant Development 

Crops were successfully harvested over five seasons. A summary of planting dates, 
flowering dates and yields are shown in Table 6.2. Planting dates ranged from 12th 



149 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



May to 25th June and yields varied between 451 and 728 gm-2. No clear correlation 
was observed between planting date and final yield. However, yields data needed to 
be made with caution as the trials differed in their location, planting density, soil fertility 
and harvest dates and as expected weather conditions varied between seasons 
(Appendix 4). To put the observed yields into perspective, clinical trials with Sativex® 
indicated that when patients were able to self-titrate, for the symptoms of MS and 
neuropathic pain, the mean daily consumption of CBD was 25 - 40 mg day-1 (9 - 14 g 
year -1) in combination with a similar dose of THC (Wade et al., 2004, Barnes, 2006). 
This suggests that one square metre of the CBD-chemovar could have provided 
sufficient feedstock for one patient for one year (not allowing for losses during 
extraction and formulation). If approved as a prescription-only medicine, the entire UK 
demand of CBD for this medicine could theoretically have been met by a crop area of 
just a few hectares. 



Year 


2000* 


2003 


2004 


2005 


2006 


Cultivar(s) 


M11 


M16 


M16 


M16 


M16 


Planting Date 


12 th May 


11 th Jun 


25 th Jun 


23 rd Jun 


23 rd Jun 


First Flowers 


No record 


1 st Sep 


22 nd Aug 


25 th Aug 


22 nd Aug 


Harvest 


6 th Oct 


8 th Oct 


9 th Oct 


10 th Oct 


17 th Sep to 
15 th Oct* 


BRM g/sq m 


502 


598 


451 


728 


609* 


CBD % w/w 


6.0 


8.8 




7.0 


7.0* 


CBD g/sq m 


30 


53 




50 


33 



Table 6.2 Summary of Agronomic and Yield Data from field trials performed between 2000 and 
2006. *The 2000 trial was performed before commencement of this thesis, and the data is 
included for comparison. 

In all the field trials, when grown from cuttings, the CBD crop gained little in height in 
the first four weeks after transplanting. Mid-June planting appeared to be sufficiently 
early for good crop establishment and no obvious benefits were observed in planting in 
May. In each year, irrespective of weather conditions and planting date, the maximum 
rate of vegetative growth was observed in the period mid-July to end August. This is 



150 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



demonstrated in Figure 6.1, which shows the results of the weekly monitoring plant 
development in the 2006 trial. The sigmoidal growth curve is similar to that reported in 
a monecious hemp crop grown from seed (Bocsa and Karus, 1998), although flowering 
occurred much earlier in hemp as the variety had been bred to mature earlier in the 
season. 



160 
140 
120 
E 100 
H 80 
£ 60 
40 
20 




t ( 



f 



fill 



16.38 


16.32 


16.22 


16.08 


15.51 


15.27 


15.08 


14.44 


14.19 


13.53 


13.26 


12.59 


12.31 


12.04 


23,Jun 


2,Jul 


9,Jul 


16,Jul 


23,Jul 


31,Jul 


06,Aug 


13, Aug 


20,Aug 


27,Aug 


03,Sep 


1 0,Sep 


17,Sep 


24,Sep 



Daylength(Hr.Mn) 
Date 



Figure 6.1 The mean height (± SD) of G5 M16 crop as observed at weekly intervals in 2005. 
Thirty plants were measured on each occasion. Data points are shown as square symbols during 
the establishment and vegetative phase. Data points are shown as triangle during the flowering 
(generative) phase. 



Observed during 2003-2006, flowering always commenced between 22 nd August and 
1 st September (Table 6.2). As with the glasshouse crop, the increase in height almost 
totally ceased seven to fourteen days after the appearance of the first stigmas. The 
final height of the crop varied from year to year, being clearly influenced by the growing 
conditions and soil nutrition. In 2005 the final crop height was 1.3 metres but the same 
clones grown in the same location in 2006 reached 2.1 metres. This was likely due to 
marked difference in mid-summer temperatures and sunshine-hours (Appendix 3) 
experienced, the conditions in Jun and July being markedly brighter and warmer. In 
four trials CBD yield was measured. Yields varied from 30 to 53 gm" 2 . However, none 
of the trials were designed with the aim of achieving the maximum possible yield. 

In 2006 floral development was observed in detail. Table 6.3 describes the stage of 
inflorescence development and stigma senescence during the flowering period, as 
recorded weekly between August 14 th and October 8 th . The table also compares the 



151 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



pattern of development with three consecutive crops of the same cultivar when grown 
in the glasshouse. As reported in Chapter 5, the proportion of senesced stigmas within 
the inflorescence gives the most useful measure of the state of maturity. Figure 6.2 
compares the pattern, speed and duration of inflorescence development and 
maturation and showed that outdoor and indoor crops of variety G5 exhibited a very 
similar pattern. This was despite the very different day lengths and growing conditions. 
In each year's trial, floral development was rapid from mid-September into the first days 
of October, when development would cease. This cessation of growth was recorded in 
some detail in the 2006 trial. Stigma senescence was observed to have commenced 
just before 23 rd September and to have been almost complete fourteen days later. 

The 2006 outdoor crop yielded well, despite the fact that mean temperatures were 
approximately 8°C lower (16.9°C - 17.3°C, Appendix 3) than the temperatures in the 
glasshouse (25°C) during the August - September flowering period. Measured ten 
kilometres away, at the glasshouse site, daily total radiation levels in autumn 2006 
were close to those predicted for this latitude using a UK global radiation algorithm 
(Hamer, 1999). This showed total daily radiation falling from 6.8 to 3.5 MJ m" 2 d" 1 over 
the flowering period. This was slightly above that encountered by a glasshouse 
production crop, which encountered a winter minimum 3.4 MJ m" 2 d" 1 rising to a 
summer maximum 5.8 MJ rnV 1 . Even though the indoor crop received supplementary 
lighting, the glasshouse roof structure reduced the light transmission levels by 
approximately a third. The glasshouse crop also experienced a shorter period of 
daylength (12 hours) throughout the flowering period, whereas the field crop 
experience longer daylengths during the first weeks of flowering. The higher radiation 
level at the commencement of outdoor flowering may be especially pertinent. 
Glasshouse studies (Chapter 5) showed that the greatest cannabinoid yields were 
achieved in crops harvested in August. These had encountered the brightest light 
conditions of the summer solstice during the very early phase of floral development. 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



Observation Timing and Pertaining Daylength 


Development stage of both indoor and 
outdoor crops. 


Indoor Crop 


Outdoor crop 


Week in 
short 
days 


Day 
length 
(Hr.mn) 


Date 
observed 


Day 

Length 
(Hr.mn) 


Inflorescence 
Development 


Stigma Development 





12.00 


13 th Aug 


14.40 


Florets absent 


Stigmas absent 


1 


12.00 


20 th Aug 


14.16 


First florets 
formed 


First stigmas visible 


2 


12.00 


27 th Aug 


13.46 


Ranid formation 

1 \ C_4 UIU 1 Wl 1 1 1 C4 LI W 1 1 

of new florets 


Pprtile stioma 5 ? 

1 Vj 1 LI 1 \j \J LI 1 1 1 C4 

common 


3 


12.00 


3 rd Sep 


13.23 


Ranid formation 
of new florets 


Prolifir ^tioma 

1 1 Ul 1 1 1 \s O LI y 1 1 1 Q 

formation 


4 


12.00 


10 th Sep 


12.56 


Ranid formation 

1 \C4 IJIVJ 1 Wl 1 1 1 C4 LI W 1 1 

of new florets 


Prolific: stioma 

1 1 Wl 1 II U OLIVJI 1 1 C4 

formation 


5 


12.00 


17 th Sep 


12.28 


Floret formation 
slows 


Fewer fertile stigmas 

observed. Older 
stigmas senesced. 


6 


12.00 


24 th Sep 


12.01 


Development 
almost ceased 


Rapid stigma 
senescence 


7 


12.00 


1 st Oct 


11.34 


Development 
ceased 


Senescence near 
complete 


8 


12.00 


8 th Oct 


11.11 


Development 
ceased 


Senescence complete 



Table 6.3 A comparison of the pattern of inflorescence development and stigma senescence in 
indoor and outdoor crops of cannabis chemovar G5. The stages of development of the 
glasshouse crop are shown from the point at which the plants are moved into a 12hour 
light/ 12hour dark until they are routinely harvested eight weeks later. The field crop 
development is shown from mid-August, just before stigma formation commenced. 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



CD 

o 
c 

CD 

o 
to 

CD 

c 

(1) 

CO 
CO 

E 

' 

CO 



100 
80 
60 
40 
20 




□ Glasshouse 
■ Field 



4 


5 


6 


7 


8 




10th Sep 


17th Sep 


24th Sep 


1st Oct 


8th Oct 


15th Oct 



Weeks in Flower (Glasshouse) 
Assessment Date (Field) 



Figure 6.2 A comparison of the pattern of stigma senescence (%, ± SD, n = 7) in 2006 field trial 
plants between 10 th September and 15 th October with that observed in five consecutively grown 
routine indoor crops of the same variety (± SD, n = 5). 



6.7.2 Comparison of the secondary metabolite yield and profile of fresh 
plant materials and enriched trichome preparations made from them 

The 2005 trial incorporated a study to compare the essential oil profile of steam distilled 
fresh mature cannabis and steam distilled ETP made from the same material. An 
essential oil fraction, produced by steam distilling foliar and floral material from ten 
plants taken at random from the crop on October 4th, yielded 7.7 ml m" 2 (7.0 g m" 2 ) The 
approximate CBD potency of plants analysed from the same crop was 7% w/w, 
indicating a CBD yield of 50 g m" 2 (500 kg ha" 1 ). It is useful to note that the essential 
oil:CBD ratio was therefore approximately 1 :7 (w/w). 

The previous day (Oct 3 rd ) ETP was made from fresh G5 M16 plant material collected 
from the same field trial. A similar batch was made from glasshouse plants at the same 
state of maturity. These were immediately frozen and steam distilled alongside the 
fresh plant material. The essential oils produced were analysed by GC at Botanix Ltd. 
The results are shown in Table 6.4. The very similar terpene profiles of the fresh plant 
material, and the ETP made from it, suggested that the complete range of 
monoterpenes and sesquiterpenes had been equally well captured during the making 
of the enriched trichome preparation. If the results of this single test were to be 
routinely replicated, this would indicate that the terpene profile of a fresh cannabis 
sample could be fairly assessed by studying an ETP made from that material. ETPs 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



made in this way are much more conveniently stored pending analysis. Being more 
concentrated they enable more minor constituents to be quantified, as demonstrated by 
trans-nerolidol in this test. Steam distillation further concentrates these terpenes. 





Field 






FrpQh 1 ppf pnrl 

l l Col l LCdl al ivj 

Flower 


Pnriphprl Triphnmp 

Preparation 


Fnrirhprl Trirhnmp 

1 1 II IUI 1 1 1 IUI 1 1 v3 

Preparation 


a-pinene 


7.7 


7.5 


9.9 


3-pinene 


4.6 


4.4 


4.2 


Myrcene 


53.2 


41.9 


38.4 


Limonene 


6.9 


7.9 


8.3 


(3-ocimene 


13.4 


13.0 


4.7 


Linalol 


1.5 


1.1 


2.6 


a caryophyllene 


2.6 


4.3 


5.4 


P caryophyllene 


10.3 


17.0 


22.7 


Trans-Nerolidol 


<1.0 


3.1 


1.4 



Table 6.4 A comparison of the terpene profile of freshly harvested fully mature field grown 
cannabis leaf and flower material (cultivar G5 Ml 6) and ETP made from the same fresh 
material (2005 Field Trial). Also included is ETP made from similar mature glasshouse grown 
material. The data show the relative peak areas when assessed by GC at Botanix Ltd. In each 
case one batch of material was analysed. 

The terpene profiles were seen to be dominated by the monoterpene myrcene and the 
sesquiterpene trans-caryophyllene. Together they accounted for at least 60% of the 
total peak area. The limited data looking at the cannabinoid, myrcene and trans- 
caryophyllene ratios of cannabis inflorescences, and the enriched trichome 
preparations made from them, indicated that the ratios were maintained during the 
making of these preparations (Table 6.4). If enriched trichome preparations could be 
made in bulk from freshly harvested field grown crops, they would avoid the 
requirement for crop drying. They would also provide a greatly enriched feedstock for 
drug production. A similar process is sometimes adopted in the hop processing 
industry. Hop glandular trichomes (known as lupulin) are collected by agitating and 
sieving deep-frozen material. 

It is interesting to note that the essential oil yield of 7.7 ml m" 2 achieved here is much 
higher than the 1.8 ml m" 2 previously recorded from unpollinated Cannabis sativa 
(Meier and Mediavilla, 1998). 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



6.7.3 The effects of harvest timing and growth stage on yield and 
cannabinoid profile 

6.7.3. 1 Botanical Raw Material Yield 

G5 was believed to be a uniquely high-yielding high-CBD chemovar, derived from 
Turkish parents naturalised at 41° N. When grown in the UK at 51° N this commenced 
flowering in the fourth week of August, in response to a phytochrome-mediated 
hormone switch, when the day-length was about 14 hrs:30 mns. At 41° N, this day- 
length would have occurred in the last week of July. This suggests that, if grown in 
Turkey, flowering would have commenced three weeks earlier. However, by the last 
week of September, the day-length would have been 12 h in both locations. Research 
findings described in Chapter 5 indicated that phytochrome-controlled systems also 
appear to induce the cessation of floral development. Floral development would 
therefore have probably ceased in both locations around the time of the autumn 
equinox at the end of September. Outdoor growing of this variety might be expected to 
be more successful if G5 production was moved closer to 41° N. The flowering period 
would be extended and conditions would likely be less conducive to the proliferation of 
Botrytis. However, the logistics of importing the crop would add to licensing difficulties 
and costs. 

In the 2006 trial, where five harvesting dates were compared (Figure 6.3), there was no 
significant difference in the yields of raw material harvested weekly between 17 th 
September and 15 th October (ANOVA). This appeared to be at least in part due to 
early-harvested crops having a higher proportion of foliar material. Later-harvested 
crops had a higher proportion of floral material but crop weight did not show a 
corresponding increase. Much of the foliage in the older crops had senesced and 
abscised by the time of harvest. It is likely that many of the primary metabolites within 
the older foliage had been translocated to the developing inflorescences in the last 
weeks of growth. 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



700 n 

600 - T T 

500 T I 1 

E 400 1 I 

ST I 1 

^ 300 - 1 - 
200 1 1 

100 

] 1 1 , 1 1 , 1 1 , 1 1 , 1 

17th Sep 24th Sep 1st Oct 8th Oct 15th Oct 

Harvest Date 



Figure 6.3 Yield of Botanical Raw Material in the 2006 trial showing the effect of planting date 
and harvest date. The results are the mean dry weights (gm~ 2 ± SD) harvested from seven 
replicates. 

6.7.3.2 CBD Concentration 

Data comparing the CBD content of crops harvested between 2000 and 2005 showed 
a range of approximately 6-9% w/w. The 2006 trial studied the effect of harvest date on 
potency, and there was a clear linear upward trend from an initial content of 2.3% to 
7.0% w/w (Figure 6.4). Analyses of variance showed highly significant weekly 
increases in potency between 24 th September and 8 th October (p < 0.01). A regression 
calculation, examining the correlation between days in flower and % CBD content 
throughout the five assessments gave a highly significant upward linear trend, 
p= 0.0028, R 2 = 0.983. 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 




17th Sep 24th Sep 1st Oct 

Harvest Date 



8th Oct 



1 5th Oct 



Figure 6.4 The effect of harvest date on cannabidiol concentration in Botanical Raw Material in 
the 2006 trial. The results are the mean % CBD (± SD) content of samples from each of the 
seven replicates, as measured by GC. Regression model, n = 7, p = 0.0028, R 2 = 0.983. 



6.7.3.3 CBD Yield 



The yield of CBD, expressed as gm , is shown in Figure 6.5. A highly significant linear 
upward trend is observed from 8gm" 2 on 17 th September to 33g m" 2 on October 15 th . 
(Regression p = 0.001 1 , R 2 = 0.994). 



40 

35 



y = 6.01 55x + 2.4709 




17th Sep 24th Sep 1st Oct 

Harvest Date 



8th Oct 



15th Oct 



Figure 6.5 The yield of CBD in the 2006 trial, showing the effect of harvest date. The results 
are the mean CBD yields (gm 2 ± SD) produced in each of the seven replicates. Regression 
model, p = 0.001 1, R 2 = 0.994 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



Despite there being no clear increase in the dry matter yield from the mid-flowering 
phase onwards, the 2006 trial showed cannabinoid yield increasing significantly in the 
first two weeks of October (ANOVA, p < 0.01). Of the acetyl CoA, NADPH and ATP 
generated by photosynthesis during this phase of growth, the plant appeared to be 
diverting a greater proportion to secondary metabolite biosynthesis. Part of the slowing 
in biomass increase during the flowering period is possibly due to the higher energy 
requirement for the production of floral tissue. Hemp crops were calculated to have a 
radiation efficiency of between 2.0 and 2.3 g/MJ PAR for vegetative growth, reducing to 
between 0.6-1.2 g/MJ PAR after flowering (Struik et al., 2000). The biosynthesis of the 
cannabinoids and terpenes would have a high energy requirement per unit weight. The 
energy required to biosynthesise the monoterpenes myrcene, pinene and limonene 
(Ci H 16 ) has been calculated to be a factor of 3.5 greater than that required for the 
same weight of glucose (Gershenzon, 1994). Although the energy requirement for 
CBDA and THCA biosynthesis is not known, metabolites with a similar C:H:0 ratio 
have an slightly lower energy cost than the monoterpenes, due to the lower state of 
reduction. Additional energy would be utilized in the formation of the complex trichome 
structure, which contains cellulose, cutin and a variety of enzyme proteins. Secondary 
metabolites formed in the last days before harvest may have been increasingly 
synthesised using metabolites released and translocated from older rapidly senescing 
tissue. 

Although the cannabinoid yield showed a continuous and significant increase up until 
the final harvest date in mid-October (p = 0.0011), the quality of the material 
deteriorated markedly as a result of fungal infection. Removal of all substandard 
material would have negated much of this yield increase. 

6.7.3.4. Effect of Harvest Date and Growth Stage at Harvest on Cannabinoid Profile 

Analysis of the BRM by GC from the earliest harvest timings showed CBD to be the 
only cannabinoid present in detectable quantities in plants of the G5 chemovar. 
Meaningful comparison of cannabinoid profiles was only achievable by studying the 
content of the much more potent enriched trichome preparations made at each 
harvest-timing. CBD, THC and CBG were present in measurable quantities in all 
samples. The ratios of these are shown in Table 6.5. 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



Harvest Date 2006 


17 th Sep 


24 th Sep 


1 st Oct 


8 th Oct 


15 th Oct 


CBG as % of 
CBG+CBD (± SD) 


5.61 ± 0.11 


5.31 ± 0.12 


3.30 ±0.18 


2.31 ± 0.12 


1.59 ± 
0.01 


THC as % of 
THC+CBD (± SD) 


4.35 ±0.20 


4.27 ± 0.13 


4.08 ± 0.11 


3.93 ± 0.04 


3.85 ± 
0.01 



Table 6.5 The changing proportions of CBG and THC with respect to CBD (Mean ± sd) in 
enriched trichome preparations produced from plants harvested at weekly intervals between 17 th 
September and 15 th October. Just one ETP sample was made on each date after bulking together 
one plant from each of the seven replicates. Three subsamples of each preparation were 
analysed. 



Results showed the importance of harvesting the crop at the correct time to achieve the 
desired cannabinoid profile. Table 6.5 showed that the CBG:CBD ratio fell rapidly 
during the last weeks of growth, the CBG proportion of the CBG + CBD total 
decreasing steadily from 5.6% on September 17 th to 1.6% on October 15th. The final 
value was close to that typically found in glasshouse crops at harvest. Forty seven 
consecutively processed batches of glasshouse-grown high-CBD chemovar were 
analysed as part of the routine growing operation. The mean proportion of CBG in the 
CBG + CBD total was 1.78% ± 0.21% SD. CBG is a cannabinoid with known 
pharmacological activity (Formukong et at., 1988). Excessive quantities of this minor 
cannabinoid in a CBD-based medicine would result in batches of feedstock being 
rejected as having an unacceptable specification. To achieve the same low CBG:CBD 
ratio as the glasshouse crop, the results of the 2006 trial suggested that the outdoor 
harvest would have had to have been delayed until mid-October. However, Botrytis 
rendered the quality of this crop unacceptable before this date. The THC:CBD ratio 
also showed a downward but less pronounced trend over the harvest period. In the 
above-mentioned batches of glasshouse-grown CBD chemovar the mean proportion of 
THC, expressed as a percentage of the THC+CBD total, was 3.89 ± 0.16% SD 
(n = 47). This is very close to the ratio found in the field grown plants in the later 
harvests (3.85 ± 0.01, Table 6.5). Homozygous high-CBD chemovar plants like G5 
produce only CBD synthase and no THC synthase (de Meijer et at., 2003). CBD 
synthase however always appears to produce a small amount of THC, as well as CBD. 
It is postulated that CBD synthase is present within CBD chemotypes in more than one 
isoform, and these variably affect the final THC:CBD ratio. The ratio of these two 
cannabinoids in commercial hemp varieties is reported to be typically around 1:20 
(Mechtler et at., 2004, Hillig and Mahlberg, 2004). The parental lines used in the 
breeding of G5 were selected on the basis of several characteristics including a 



160 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



THC:CBD ratio as low as possible. As a consequence, the ratio in G5 plants was lower 
than most commercial hemp varieties (deMeijer, pers comm), decreasing from 1:23 in 
the early harvest to 1:26 over subsequent weeks (tentative n = 1 data only). This 
apparent decrease in later harvested plants was possibly due to isoforms of CBDA 
synthase being present which differed in their ability to function in the increasingly 
autumnal growing conditions. However despite this, as the CBD content rose to 5.8 % 
and 7.0 % w/w in the final weeks (Figure 6.4), the corresponding THC content rose to 
0.23 % and 0.27 %. 

As designated under the Misuse of Drugs Act Cannabis sativa can be licensed only for 
research or "other special purposes" providing the Secretary of State is of the opinion 
that it is in the public interest to do so. The only "special purpose" currently recognised 
is the cultivation of EU-approved varieties of hemp for commercial and industrial 
purposes. All such EU-approved varieties have THC contents below 0.2%. When this 
program of field trials commenced, the maximum THC content permitted in floral 
samples of licensed hemp crops was 0.3% w/w. This was subsequently reduced by 
the European Union (EU regulations (VO (EG) Nr. 1420/98)) to 0.2%. Although a 
minimum content of 0.3% was readily achievable by chemovar G5, the lower limit of 
0.2% may be too low for the G5 chemovar to qualify for an EU subsidy payment, and 
this would have a small effect on the cost of outdoor growing. Growing a variety 
containing more than 0.2% THC is not illegal, but does necessitate a dispensation from 
the Home Office Licensing. Enquiries to the Home Office (Drugs Licensing) indicate 
that such an application would be met favourably (Evans, 2008). 

6.7.4 Effect of Growth Stage and Harvest Date on Essential Oil Profile 

The 2006 trial compared of the terpene profiles of field grown cannabis plants 
harvested at weekly intervals between 17 th September and 15 th October. This was 
achieved by first steam distilling an enriched trichome preparation made from fresh 
botanical raw material collected on each date. Between 30 ml and 100ml of enriched 
trichome preparation was produced from each, but this contained a large unmeasured 
volume of residual water. It is not possible to quantify the essential oil concentration of 
each sample on the basis of the non-aqueous fraction only. When distilled these 
produced between 0.25 ml and 2.1 ml of essential oil and each was analysed by GW 
Pharmaceuticals using GC. The relative peak areas for the twenty most abundant 
terpenes detected are shown in Table 6.6. These twenty accounted for more than 97% 
of the total peak areas. The monoterpene:sesquiterpene ratio of each of the essential 
oil samples showed a weekly increase. A regression calculation showed the weekly 
linear increase in the proportion of monoterpenes to be strongly correlated with time (p 



161 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



= 0.0052, R 2 = 0.973, y = 0.844x + 0.6219). All individual sesquiterpenes, of which 
trans-caryophylene was the most dominant, showed a decreasing presence in the 
overall terpenoids mixture. 

The data show that of the monoterpenes, myrcene was by far the most dominant. It 
was notable that when expressed as a % of the total peak area, the proportion of 
myrcene more than doubled over the sampling period. Limonene and beta-ocimene 
also showed clear though less dramatic increases. The pinenes conversely showed a 
possible downward trend. 



162 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 





Harvest Date 2006 




17 th Sep 


24 th Sep 


1 st Oct 


8 th Oct 


15 th Oct 




% of Peak Area 


Monoterpenes 












Alpha-pinene R 


3.49 


5.27 


4.25 


3.13 


3.24 


Alpha-pinene S 


4.30 


2.35 


3.63 


3.78 


3.00 


Beta-pinene 


3.32 


3.50 


3.33 


3.18 


3.00 


Beta-Myrcene 


22.63 


36.30 


40.86 


46.02 


51.48 


Limonene 


2.75 


3.91 


4.31 


4.68 


5.07 


Beta-ocimene 


2.64 


4.13 


4.04 


4.63 


4.76 


Sesquiterpenes 












t-Caryophyllene 


37.44 


29.10 


26.57 


23.97 


20.17 


Bergotamene 


0.34 


0.18 


0.16 


0.12 


0.08 


Humulene 


11.31 


8.57 


7.60 


6.71 


5.67 


Aromadendrene 


0.67 


0.34 


0.31 


0.19 


0.14 


Selinene 


1.17 


0.63 


0.52 


0.36 


0.29 


Anon 


0.84 


0.46 


0.41 


0.26 


0.21 


Z,E Farnesene 


0.18 


0.09 


0.08 


0.06 


0.07 


alpha farnesene 


1.70 


1.77 


1.44 


1.25 


1.11 


alpha Gurjunene 


0.17 


0.15 


0.11 


0.09 


0.07 


Bisabolene 


0.78 


0.42 


0.37 


0.22 


0.16 


Nerolidol 


0.66 


0.62 


0.38 


0.26 


0.23 


Caryophyllene Oxide 


4.13 


1.70 


1.29 


0.91 


1.05 


Diepicedrene-1 -oxide 


0.89 


0.38 


0.28 


0.17 


0.19 


Alpha-Bisabolol 


0.59 


0.12 


0.07 


0.02 


0.02 


Total Monoterpenes 


39.12 


55.46 


60.41 


65.42 


70.55 


Total Sesquiterpenes 


60.88 


44.55 


39.59 


34.58 


29.45 



Table 6.6 A comparison of the terpene profile of steam-distillates of enriched trichome 
preparations made from freshly harvested plants on five dates between 17 th September and 15 th 
October 2006. One bulked sample was analysed on each date. 



Although the relative peak area data in Table 6.6 show very clear changing ratios 
between individual terpenes, and between the overall ratios of monoterpenes to 
sesquiterpenes, the true content of each of the twenty terpenes could not be accurately 
gained from this data. Quantitive data was generated for eight terpenes for which 
analytical standards were readily available. The results are shown in Table 6.7. 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



Harvest Date 


17 th Sep 


24 th Sep 


1 st Oct 


8 th Oct 


15 th Oct 


a-Pinene 


9.18 


8.44 


7.82 


7.09 


6.18 


(3-Myrcene 


40.45 


58.16 


61.00 


70.01 


74.30 


Limonene 


2.89 


2.61 


3.84 


2.82 


2.84 


Linalool 


0.40 


0.60 


0.35 


0.13 


0.26 


trans- 
Caryophyllene 


32.36 


21.60 


19.52 


15.37 


12.68 


a-caryophyllene 


9.18 


5.93 


5.27 


4.02 


3.30 


Caryophyllene 
Oxide 


4.14 


1.52 


1.46 


0.02 


0.03 


t-Nerolidol 


1.39 


1.13 


0.74 


0.53 


0.41 


Ratio Myrcene/ 
t-caryophyllene 


1.25 


2.69 


3.13 


4.55 


5.86 



Table 6.7 Ratio of eight terpenes in steam distilled enriched trichome preparations made from 
freshly harvested field grown plants of cultivar G5 Ml 6. The result for each terpene is 
expressed as a weight percentage (% w/w) of the total within each column. The table also 
shows the ratio of myrcene (the dominant monoterpene) and trans-caryophyllene (the dominant 
sesquiterpene). 

The qualitative peak area data in Table 6.6 gives a valuable though not accurate 
measure of the ratios between the individual terpenes. Comparing quantitative data, as 
in Table 6.7, is more accurate but limited by the availability of reference standards. 
The two analytical methods are compared in Table 6.8, which shows the 
myrcene/trans-caryophyllene ratios when calculated from Relative Peak Area and 
quantitative w/w data. Each is expressed as a % of the October 15 th value. Whether 
determined from the peak area data or the w/w data, the 'normalised' values give very 
similar results with the myrcene/trans-caryophyllene ratio doubling between September 
24 th and October 15 th . 



164 



Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



Harvest 
Date 


17 th Sep 


24 th Sep 


1 st Oct 


8 th Oct 


15 th Oct 




Myrcene/t-caryophylene 




Actual Value 


Peak Area 


0.60 


1.25 


1.54 


1.92 


2.55 


w/w 


1.25 


2.69 


3.13 


4.55 


5.86 




Normalized Value Shown as % of 15 th Oct Value 


Peak Area 


24 


49 


60 


75 


100 


w/w 


21 


46 


53 


78 


100 



Table 6.8 Comparison of myrcene/trans-caryophyllene ratios when calculated from Relative 
Peak Areavalues and w/w data. 



6.7.5 Comparison of the secondary metabolite content of glasshouse and 
outdoor grown plants 

As reported above in Section 6.7.6, a single field trial suggested that the THC:CBD 
ratio of late harvested G5 CBD-chemovar plants was similar if plants were grown in 
either environment. A more dramatic effect of outdoor growing was demonstrated by 
five heterozygous 6 T 6 D genotypes derived from variety G159. These produced a 
significantly lower mean THC:CBD ratio when grown outdoors (two-tailed t-test, p < 
0.001, Table 6.9). 





THC as % of THC+CBD (n = 4) 


Clone 


G 159/9 


G 159/11 


G 159/1 2 


G 159/1 3 


G 159/1 6 


Mean 


Field 


33.1 ±2.4 


33.2 ± 0.2 


48.9 ± 1.9 


33.9 ±0.0 


33.3 ±0.1 


36.5 


Glasshouse 


38.8 ± 1.5 


39.9 ±0.6 


57.4 ± 0.9 


40.6 ± 0.6 


38.6 ±2.0 


43.1 



Table 6.9 The relative proportions of THC and CBD synthesised in heterozygous B T B D clones 
derived from variety G159. The proportion of THC produced is shown as a % of the 
THC+CBD total. Just one sample of dry inflorescence material was analysed from each plant. 

In a separate study not reported here, where these clones were grown in a growth 
room in identical conditions apart from contrasting growth temperature of 15°C and 
25°C, the cooler temperature significantly increased the proportion of CBD within the 
cannabinoid profile. A possible explanation is that in cooler conditions the CBD 
synthase within these plants is able to compete more favourably than THC synthase for 
the common precursor CBGA. No previous reports could be found describing this 
temperature effect. As THC synthase and CBD synthase have been suggested to exist 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



in more than one isoform (de Meijer, 2003), other heterozygous mixed THC/CBD 
chemotypes might exist which synthesise different ratios of one or both of these 
synthases. These in turn may synthesise differently to temperature. It is also possible 
that the increased proportion of CBD observed here is specifically induced by the 
activation of an unidentified gene. In either case, the ability to produce proportionally 
more CBD in cooler conditions may be an inherited trait which has at some point 
improved the survival ability of Cannabis sativa. 

Changing temperatures have been found to alter the ratios of terpenoids in glandular 
trichomes of other species e.g. Pelargonium xhortorum. Walters and Harman (1991) 
showed that growing temperature affected the ratio of C 2 2 and C 2 4 unsaturated 
anacardic acids, both of which have similar molecular weights to the cannabinoids. It 
was postulated that plants may be exhibiting a genetically controlled response to 
temperature by altering the proportion of less viscous secondary metabolites within the 
trichome, to maintain uniform viscosity. A potential benefit of doing so would be the 
retention of the trichome's ability to ensnare insects. Although the effects of 
cannabinoid profile on trichome viscosity have not been tested, this explanation 
seemed unlikely in cannabis. At room temperatures THCA is an oil and CBDA is a 
crystalline solid. To retain stable viscosity as temperatures fell, it seems probable that 
the trichome contents would require proportionally more of the oil. Observations in this 
study demonstrated that the opposite had occurred. The cannabinoid profile may have 
been affected by the higher proportion of UV light encountered outdoors. This part of 
the spectrum is less able to penetrate a glasshouse and the amount produced by HPS 
lamps is negligible. However, contrary to the proportional decrease in THC synthesis 
observed in this outdoor crop, Lydon et al. (1987) showed that UV B radiation 
significantly increased THC biosynthesis, whereas CBD biosynthesis did not show a 
significant increase. However, Lydon et al. (1987) did not show the actual CBD data 
and did not include a test of the statistical significance of the difference in biosynthesis 
of THC and CBD when exposed to increasing UV B radiation. 

To facilitate the comparison of the terpene profile of indoor and outdoor grown plants of 
cultivar G5 M16, the mean peak area for each of the terpenes was calculated with each 
of the five sampling dates being regarded as a single replicate. The results are shown 
in Figure 6.6. To allow a clearer comparison between the contents of both major and 
minor terpenes, the results are expressed as % of Total Peak Area using a logarithmic 
scale. The terpene profile of these indoor and outdoor grown plants was seen to be 
very similar, with no significant difference in mean % peak area in eight of the fourteen 
terpenes (p > 0.05). Being based upon one indoor and one outdoor crop the relevance 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



of these differences has to be viewed with caution. The terpenes showing the greatest 
significant difference in content were ocimene and nerilidol, which had proportionally 
lower contents in outdoor-grown plants. This may have been a result of higher UV 
light conditions outdoors. UV B radiation has been found to alter terpene profiles in other 
species e.g. basil Ocimum basilicum (Johnson et al., 1999), thereby affecting the 
flavour and commercial value of this culinary herb, but no obvious survival advantage 
was attributed to this terpene profile change. The reason for the altered terpene profile 
in outdoor grown cannabis plants may alternatively be a phytochrome-mediated 
response, as observed in outdoor grown thyme, Thymus vulgaris (Tanaka et al., 1989). 
Many of the terpenes found in glandular trichomes have been found to be repellant to 
specific insects, and there may be an inherited benefit in only biosynthesizing certain 
secondary metabolites to coincide with the life-cycle of a major predator. 



100.0 n 




Terpene 
(ANOVA) 



Figure 6.6 A comparison of the terpene profiles, as a proportion of the total peak area, of 
enriched trichome preparations prepared from glasshouse and outdoor crops (f = Caryophyllene 
Oxide). The results are the mean of five samples produced at weekly intervals towards the end 
of flowering (± SD). Glasshouse plants had been in a 12 hour day length for 6 to 10 weeks. 
Field grown crops were sampled between September 17 th and October 15 th . 
(ANOVA, Glasshouse v Field, * p < 0.05, ** p < 0.01, *** p < 0.001). 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



6.7.6 Summary of Pest and Disease Problems in the Field Trials 

The main pest and disease problems incurred are summarised in decreasing order of 
severity in Table 6.10. 





Severity Score 1 -5 
(1 -absent, 2-minor, 3-moderate, 4-severe, 5-fatal) 




^^^^^ Year; - 
Pest ^^^^ 


ZUUU 


ZUUO 




ZUUO 


ZUUO 


orey iviouiq 
Botrytis cinerea 


1 


3 


3 


3 


1-5* 


Caterpillars (Various spp) 


2 


3 


3 


3 


2 


Stemphylium Leaf Spot 


1 


1 


1 


1 


3 


Common Nettle Capsid f 
Liocoris tripustulatus 








2 


2 


Leaf Miner 

Liriomyza strigata 


1 


1 


1 


1 


2 


Powdery Mildew 
Sphaerotheca macularis 


1 


1 


1 


1 


2 


Black Bean Aphid 
Aphis fabae 


1 


1 


1 


1 


11 



Table 6.10 Summary of pest problems experienced, in decreasing order of magnitude using a 
subjective 1-5 score. * Botrytis initially minor but extremely severe in late harvested plots, 
f Symptoms not recognized as pest damage until 2005. $ Aphids absent in trial plots but 
moderate infestation of black bean aphid Aphis fabae observed on neighbouring cannabis seed- 
crop with lower secondary metabolite content. 



Grey Mould Botrytis cinerea was observed in all but the first year's trial. The disease 
regularly attacked the inflorescence tissue of mature plants (Fig 6.7) but was never 
observed on the floral tissue of G5 plants before 1 st October. Botrytis is reported to be 
the most common disease of cannabis, proliferating in high humidity which encourages 
conidia germination (McPartland at al., 2000). The disease would typically establish 
deep within the inflorescence, where the humidity was especially high. In many cases 
the disease was only discovered by manually parting the bracts. In the 2006 field trial 
the level of B. cinerea was assessed on plants as they were harvested on five harvest 
dates between 18 th September and 17 th October. On each occasion the number of 
plants visibly infected in each plot was recorded. The results are shown in Table 6.11. 
The first infected plants were observed on 2 nd October. Although 12% of plants were 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



visibly spoiled, only a small proportion of each plant was affected. To comply with 
Paragraph 3.1 of the GAP Guidelines the harvest should take place when the plants 
are of the best possible quality according to the different utilizations. If harvested at this 
stage, the crop would have to be laboriously graded to remove the poor quality 
diseased material. By the 8 th October a large proportion of the crop was affected and 
grading the crop to only retain high quality material would have been especially difficult. 
A week later on 17 th October almost all of the plants were affected and the crop was 
regarded totally unacceptable as a phytopharmaceutical feedstock. 




Figure 6.7 Fungal damage of a cannabis inflorescence due to Botrytis cinerea. 



Harvest Date 


17 th Sep 


24 th Sep 


1 st Oct 


8 th Oct 


15 th Oct 


% of Plants Infected 








12.2 


30.6 


98.0 



Table 6.11 The level of infection with Botyrytis cinerea observed on plants harvested on five 
dates between 17 th September and 15 th October 2006. Scores are the mean % infection rates in 
the seven plots of each treatment 

Other diseases experienced were Stemphylium Leaf and Stem Spot (Pleospora tarda 
E.G. Simmons) and powdery mildew (Sphaerotheca macularis). Stemphyllium has 
been recorded as a significant problem in hemp crops in Canada, Holland and Eastern 
Europe (McPartland et al., 2000). In the field trial program the disease was only 
observed in 2006. The identification was made by CABI Bioscience, Bakeham Lane, 
Egham, Surrey, TW20 9TY, UK. The fungus was reported to be in its anamorphic state 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



- Stemphylium botryosum Wallr. Symptoms became widespread on foliage in August. 
The discoloured leaf tissue within the spot necrosed and frequently disintegrated to 
leave a characteristic 'shot hole' in the leaf. Towards late September these spots 
would coalesce leading to a significant loss of leaf area. Throughout this latter phase 
however, the bract tissue within the inflorescences was totally unaffected by the 
disease. 

Powdery mildew appeared in a field trial for the first time in 2006 as a minor disease. 
No plants could be regarded as being of insufficient quality for harvest. Of the insect 
pests to attack the crop caterpillars (various species) were present each year and 
caused greatest damage, especially in late summer. However, damage was generally 
restricted to the large leaves that subtended the top inflorescence. In each case the 
capitate stalked trichomes on the bracts and bracteoles proved an excellent deterrent 
to predation, as shown in Figure 6.9. Active caterpillars were often discovered within 
the inflorescences but were observed not to be eating the resinous bracts and 
bracteoles. 




Figure 6.8 A cannabis plant at the late flowering stage. Resinous bracts are unaffected but 
leaves below the inflorescence are heavily grazed. In some cases little more than the midrib of 
the leaf remains. 

Other insects pests observed included leaf miner Liriomyza (Agromyza) strigata 
(Meigen), black bean aphid Aphis fabae and common nettle aphid (Liocoris 
tripustulatus). In no year was more than an estimated 2% of the foliage lost to leaf 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



miner, the problem being most severe in 2006 when prolonged higher-than-average 
temperatures favoured attack. Aphids were notable for their total absence in all seven 
year's crops of the G5 CBD chemovar. However, the pest was noted in 2006 on a 
susceptible cannabis variety planted from seed alongside the G5 CBD trial. An 
analysis of variance showed that the males were significantly more susceptible than 
the females (males 11.9%, females 1.3%, p < 0.001). Cannabis appears therefore to 
be typical of so many dioecious crops where the male is more susceptible to predators 
- a clear example of sex-biased herbivory (Herms and Matson, 1992). The all-female 
G5 crop appeared to be benefitting from both a varietal and sex-based resistance to 
Aphis fabae. 

Common nettle capsid is reported to feed on common nettle (Urtica spp.) and to 
occasionally cause economic damage in a number of glasshouse crops (Malais and 
Ravensberg, 1992). This was observed on regular occasions in outdoor crops during 
August when larvae from the summer generation have reached the adult stage. 
Commercial fibre and seed-hemp crops would typically be approaching their natural 
harvest date in August and be beyond a stage at which serious damage could occur. 
However, the all-female cannabis drug-crop would be producing ample succulent 
foliage at this time and clearly more susceptible to attack. Also encountered but not 
quantified were rabbits and arable weeds. After excessive grazing damage in 
2001/2002 all subsequent trials were protected with rabbit proof fence. Weeds were 
manually removed at intervals through the season. 

6.7.7 Effect of Raised Temperatures on Crop Drying Time 

The initial attempt to dry cannabis plants on the floor of an oast house drying chamber 
was only partially successful. To maintain a uniform rate of drying through a hop crop, 
the material needs to be spread evenly on the floor in sufficient depth to substantially 
restrict air flow. Such a crop is typically dried within a few hours. The depth of the 
cannabis (approximately 15 cm) was much less than the one metre depth of hops 
traditionally processed in the oast, and the inlet temperature of 40°C was at the lower 
end of the typical range (Neve, 1991). To successfully dry the crop this way required 
twenty four hours. 

The crops in 2004-2006 were hung to dry on wires suspended within the oast house. 
Cannabis crops typically feel sufficiently dry to be stripped when below 15% moisture. 
Figure 6.10 suggests that in 30, 40 and 50°C, the mean times taken to achieve 15% 
moisture were approximately 36, 18 and 11 hours respectively. Plants of the same 
cultivar, dried in the glasshouse-crop drying facility at 25°C typically took 4.5 - 5.0 days 
to reach this same moisture level. However, ventilation levels differed between the two 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



locations and the longer drying time cannot be entirely attributed to a lower drying 
temperature. This prolonged drying time was highly favourable to the proliferation of 
fungi and bacteria, and plants already showing low levels of disease were seen to 
deteriorate in these conditions. Increased drying temperatures greatly reduced the 
time within which such fungal spoilage could occur. It was accepted that proportionally 
more of the volatile monoterpenes would be lost compared to the sesquiterpenes at the 
higher drying temperatures. However, it was considered likely that the increased 
monoterpene losses at 40°C and 50°C were minor compared to those that would have 
been lost during the decarboxylation process, when milled BRM was heated at much 
higher temperatures to convert the cannabinoid acids into the neutral forms. 




□ 30 C 

■ 40 C 

■ 50 C 



Drying Time (Hours) 



Figure 6.9 The rate of moisture loss of field grown cannabis, when dried at three temperatures 
(30, 40 and 50°C). The results are the mean of three crops dried in 2004-2006 (± SD) and show 
the pattern of moisture loss until mean moisture content was <15%. 

6.8 CONCLUSIONS 

The research reported here indicated for the first time that the outdoor propagation of 
Cannabis as a phytopharmaceutical in the UK was possible. However, the largest 
problems to overcome were disease control and the logistical difficulties encountered in 
harvesting a large volume of crop in a limited period of time. Cannabinoid and terpene 
profiles of indoor and outdoor gown crops were shown to be very similar if both crops 
were harvested at the end of floral development, as denoted by complete stigma 



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Chapter 6. The Outdoor Propagation of Phytopharmaceutical Cannabis 



senescence. However, the desired cannabinoid profile was only achieved in mid- 
October, when levels of Botrytis infection were high. The reliable outdoor production of 
this crop appears impossible therefore without the use of fungicides. This would 
necessitate harvested crops being tested for fungicide residues. 

The cannabinoid profile of the outdoor crop appeared less stable than that of crops 
grown in the glasshouse. As a result, the harvest would need to take place within a 
very short period when cannabinoid levels were assessed as being within the agreed 
specification. This could coincide with unfavourable weather conditions, which would 
hamper harvest operations. However, the ability to rapidly dry the crop was 
demonstrated. An alternative way of exploiting outdoor grown Cannabis may be to 
collect the glandular trichomes from this material. The crop could conceivably be 
harvested and promptly fresh frozen for processing at a later date. Similar harvest 
operations have been used in the frozen pea industry for decades. As stated in the 
previous chapter, the bulk removal of Humulus trichomes is also practised industrially. 
The concept may warrant evaluation in forthcoming seasons. 



173 



Chapter 7 General Discussion 



Chapter 7 General Discussion 

GW Pharmaceuticals pic, the sponsoring company, has been resolute in developing 
cannabinoid-based phytopharmaceuticals from Cannabis sativa L. At the 
commencement of this thesis in autumn 2003 the company was propagating two 
chemotypes to produce the medicine Sativex®. This was undergoing Stage III clinical 
trials for the treatment of symptoms of multiple sclerosis (MS) and for various forms of 
pain relief. In Canada in 2005 Sativex® received provisional approval with conditions for 
the treatment of central neuropathic pain in MS, and in 2007 for intractable cancer pain. 
In 2008 GW Pharmaceuticals commenced Stage III clinical trials in the US to evaluate 
the efficacy of this medicine for the control of pain in terminal cancer patients. Since 
2005 Sativex® has been available in the UK on a 'named-patient' basis, with the Home 
Office being notified of each recipient. Many other countries have since allowed 
prescription of the medicine on the same basis. The medicine contains two major 
cannabinoids, A 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD), which both had 
reported pharmacological properties. The choice of these two cannabinoids was based 
upon evidence that, in addition to them individually being pharmacologically active, the 
two acted together synergistically, with CBD abrogating the side effects of anxiety and 
intoxication associated with THC alone (Fowler and Law 2006). Sativex® was a 
botanical drug, i.e. a well characterised, multi-component standardised drug extracted 
from plant sources. In addition to THC and CBD, the medicine also contained lesser 
amounts of other cannabinoids, as well as monoterpenes and sesquiterpenes. In vivo 
rodent studies showed cannabis extracts to have significantly increased 
pharmacological activity over cannabinoids alone, (Williamson, 2001) and the 
monoterpenes and sesquiterpenes were suspected of being at least partly responsible 
for this increase. It was envisaged that these terpenes might also prove to be 
advantageous ingredients in Sativex®. 

To meet the quality and safety standards demanded of a medicine by the Regulatory 
Authorities, the Botanical Raw Material used as the starter material for Sativex®, and 
possible future botanical medicines, had to contain a minimum and maximum level of 
each of several secondary metabolites. To increase the probability of each batch of 
botanical raw material meeting this specification, the growing conditions initially 
adopted were kept as uniform as possible and plants routinely harvested when of a 
fixed age. Initial attempts to grow year-round uniform material were thwarted by a 
seasonal fluctuation in yields. The cause of this was suspected to be seasonal variation 
in irradiance levels within the glasshouse. Relatively little was known of how alterations 



174 



Chapter 7 General Discussion 



to the growing protocol would affect the secondary metabolite profile of the harvested 
plants. It was possible therefore that any attempts to improve the growing conditions 
might produce feedstock that failed the product specification. A series of tests was 
performed to analyse the effect on plant development and secondary metabolite 
content of altering growing conditions. As part of this, seed-sown plants were tested 
alongside those derived from cuttings (clones). Plants were compared when grown in 
varying daylengths and under differing irradiance conditions. A range of harvest timings 
was also compared. 

Most if not all other phytopharmaceuticals are produced from outdoor grown crops. 
Growing cannabis indoors provided better growing conditions and higher level of 
security. However, the process was costly and produced a large carbon footprint. The 
energy consumed for lighting alone exceeded 0.5 kW hr per gram of dry feedstock for 
glasshouse crops, and reached approximately 1 kW hr per gram in a totally enclosed 
environment. Applying equally high levels of security to the growing of CBD chemovar 
crops was arguably excessive. It was recognised that outdoor growing of cannabis 
would reduce energy costs, but the effects on secondary metabolite profile and plant 
quality might be unacceptable. The vagaries of the weather would be expected to 
influence yields but how this would affect secondary metabolite profile was not known. 
Pest and disease problems would likely be different to those encountered by a 
glasshouse crop and this might unacceptably affect quality. Drying the crop would also 
present logistical difficulties. Prior to commencing the work of this thesis, one CBD crop 
had been grown successfully outdoors and the cannabinoid yields achieved were 
similar to those achieved with a single glasshouse crop. For this thesis a sequence of 
CBD-chemotype crops was grown outdoors and plant development studied. Pest and 
disease levels were monitored. Crops were harvested over a range of dates from mid- 
September to mid-October and the effect of harvest date on cannabinoid and terpene 
profile compared to that of glasshouse crops. In an attempt to reduce the degree of 
crop spoilage during the drying process, trials were performed over three seasons to 
compare crop drying regimes at a range of temperatures. 

When characterising any phytopharmaceutical feedstock, a systematic and illustrated 
report of the microscopical details is generally required. In Cannabis sativa L, the 
trichomes were perceived to be the most important parts of the plant, as the 
cannabinoids and terpenes are biosynthesised and sequestered within these 
structures. A review of the form, function and distribution of these constitutes Chapter 
Three of this thesis. As the chapter shows, there are differing trichome forms. The 
cannabinoid and terpene profile of the plant changes through its life and this can often 



175 



Chapter 7 General Discussion 



be at least partly explained by the presence of changing proportions of so-called 
sessile and capitate stalked trichomes. Most notable examples are the higher 
proportions of CBC and sesquiterpenes within the secondary metabolite profile of 
foliage compared to floral material, this being attributable to the absence of capitate 
stalked trichomes on foliage. In potent female floral material the total volume of 
secondary metabolites in sessile trichomes is almost negligible compared to that in the 
larger capitate stalked trichomes. Consequently, although they have a different profile, 
the sessile trichomes have little influence on the overall secondary metabolite profile of 
the plant. So-called 'manicuring' of inflorescence, to remove bract tissue lacking 
capitate stalked trichomes, is time consuming. Although favoured by producers of 
recreational cannabis, seeking to improve potency and flavour, the process has little 
effect on the profile of the remaining feedstock. 

The removal of glandular trichomes from cannabis floral material produces an 'enriched 
trichome preparation' that has a very similar cannabinoid and terpene profile to that of 
the plant material from which it came, but is much more potent. One major exception is 
the diterpene phytol, which is associated with the biosynthesis and catabolism of 
chlorophyll and Vitamin E, and this is predominantly found outside the trichome. Due to 
the naturally small volume of the sessile trichome population, the collection of these 
from aerial tissues produces very small quantities of material. However, the secondary 
metabolite profile of the collected material can be of great potential interest. The 
removal and sieving of sessile trichomes from aerial parts of a CBC-rich chemovar 
containing 1.4% w/w CBC produced a preparation that contained 44% w/w CBC. The 
stage of development of these sessile trichomes can greatly affect their cannabinoid 
profile. In the example just quoted CBC accounted for 61% of the total cannabinoids 
present in the raw material. However, the agitating and sieving process predominantly 
captured the older more mature sessile trichomes and the CBC purity level of the 
resultant filtrate was 94% w/w. This method of producing an enriched source of CBC, 
by selectively capturing mature sessile trichomes, was submitted for patent protection. 
Enriched trichome preparations, predominantly containing capitate stalked trichome 
resin heads, were produced which contained up to 67% w/w THC. This material has 
the clear advantage of being less bulky than feedstocks containing botanical raw 
material. It also lacks the chlorophyll-based pigments and other substances found in 
foliage, which may be regarded as undesirable in some botanical medicines. When 
considering possible ways of mechanising the removal and collection of glandular 
trichomes, it was recognised that harvesting of cannabis glandular trichomes is also the 
basis of illicit cannabis resin manufacture. The efficiency of this was assessed in 



176 



Chapter 7 General Discussion 



studies to characterise the illicit cannabis materials typically used in England in 
2005/2005. 

Resin is just one form of illicit cannabis circulating in the UK. Also commonly found are 
imported outdoor-grown 'herbal cannabis' and a more potent intensively indoor-grown 
all-female cannabis product called sinsemilla - or more colloquially 'skunk'. Illicit 
cannabis is used for recreational and medicinal purposes. Indeed, it was partly on the 
basis of increasing evidence of the efficacy of this material that GW Pharmaceuticals 
was given Home Office permission to evaluate cannabis as a phytopharmaceutical. 
However, little was known of the cannabinoid profile of this material and the impact that 
this would have on its efficacy and safety. For this thesis, the first survey of UK 
cannabis cannabinoid content was performed. 

The study showed that herbal, sinsemilla and resin contained very variable quantities 
of THC and herbal and sinsemilla cannabis were almost devoid of CBD. Resin 
contained extremely variable quantities of CBD, and along with herbal cannabis often 
contained high levels of CBN. The ratio of these cannabinoids in resin varied greatly, 
much of this due to the fact that THC catabolised much faster than CBD. In theory, a 
variable content of one cannabinoid could be overcome by patients if they self-titrated 
to achieve the desired dose level. When using resin, the only significant source of 
CBD, self-titration did not offer the ability to correctly dose both THC and CBD. 
Although the only significant source of CBD, previously unseen survey data analysed 
for this thesis showed that resin was unfavoured amongst the majority of medicinal 
cannabis users. Whereas the appearance of resin gave no indication of its likely 
cannabinoid content, simple visual organoleptic assessments did give a significant clue 
as to the THC content of sinsemilla. Part of the preference for sinsemilla amongst 
some medicinal users is the ability (albeit illegal) to grow the material at home. 
Although seed is commercially available, it is almost always of the high-THC genotype. 
The study showed that the resin's share of the illicit cannabis market was in decline, 
with sinsemilla becoming more dominant. The THC content of sinsemilla in the UK was 
showing a significant upward trend. The psychoactive potential of the average 
cannabis sample was thus increasing. This was due to the increasing THC content, 
combined with a decline in the presence of the antipsychotic cannabinoid CBD. This 
data was used by the Advisory Council on the Misuse of Drugs before making 
recommendations to the UK Government on the possible reclassification of cannabis 
(ACMD, 2008). 

Chemical characterisation involved the analysis of whole cannabis plant tissues and 
glandular trichomes collected from them. Unpollinated female floral tissues were known 



177 



Chapter 7 General Discussion 



to be the greatest source of cannabinoids and terpenes and studies were focused here. 
Most cannabis varieties showed a strong photoperiodic response and, after an initial 
few weeks of vegetative growth in continuous lighting, plants generally commenced 
flowering within ten days of being switched to a daylength of 12 hours. Inflorescences 
produced increasing numbers of florets over subsequent weeks but after eight weeks in 
short days, growth was slowing rapidly and few additional fertile stigmas were formed. 
The cessation in floral development was mirrored by a slowing in cannabinoid 
biosynthesis. Over this period the cannabinoid profile changed, with the proportion of 
CBG decreasing significantly. Outdoor grown CBD chemovars showed a very similar 
pattern of floral development and cessation of flowering was matched by the same 
decrease in the proportion of CBG in the cannabinoid profile. The terpene profile of the 
plant also changed during plant development and, whether grown in stable glasshouse 
conditions or outside, the pattern of change was similar with the profile stabilising as 
the formation of new florets ceased. 

The cannabinoid profile of other chemovars also showed a marked change during the 
flowering process. This was most pronounced in heterozygous chemovars producing 
more evenly mixed cannabinoid profiles. One of these (variety G159) was the source 
of several clones that synthesised THC and CBD in approximately equal quantities, 
and the ratio was observed to change significantly during the flowering process. These 
clones all produced a significantly higher proportion of CBD when grown outdoors in 
cooler average temperatures. Growth room tests confirmed that cooler propagation 
temperatures significantly increased the proportion of CBD within the cannabinoid 
profile. This could be simply due to the CBD synthase enzyme having a proportionally 
greater efficiency than THC synthase at lower temperatures, or to the plant switching 
on some mechanism to enhance CBD synthesis at the expense of THC. This finding 
has a wider implication for the understanding of how THC and CBD chemovars 
perhaps evolved and were exploited by man. THC-dominant chemovars are typically 
associated with latitudes of 30° or less (Small, 1976) where the plant is most capable of 
producing potent material and the local culture enables its production. CBD-dominant 
chemovars are associated with temperate latitudes, and this is heavily influenced by 
licensing restrictions that only allow the exploitation of this chemotype for fibre or seed 
production. This observation with heterozygous cannabis suggests that in a landrace 
population, which would include homozygous THC and CBD chemovars and 
heterozygous mixed THC+CBD chemovars (de Meijer, 2003), a higher proportion of 
THC would be synthesised in the warmer climates irrespective of the involvement of 
man. 



178 



Chapter 7 General Discussion 



Although the glasshouse used for this research had sophisticated environmental 
management and supplementary lighting systems, as stated earlier there was a large 
seasonal fluctuation in light conditions within the building. This was the presumed 
explanation for large seasonal variations in crop yield. Although the existing 
supplementary lighting system was capable of adding 16 W m" 2 of photosynthetically 
active radiation (PAR) to the natural light, and originally considered adequate to 
support the healthy growth of many plant species, this proved inadequate for uniform 
year-round growth of Cannabis. Winter yields of botanical raw material were 
significantly reduced and there was an even greater reduction in the yield of 
cannabinoids. Following growth room tests, this was overcome by the provision of an 
improved supplementary lighting system capable of adding photosynthetically active 
radiation levels of 53 W m" 2 to that provided naturally by daylight. This level of lighting is 
more than twice that typically used in the production of UK glasshouse-grown food- 
crops. It was discovered that high irradiance levels appear to be most essential at the 
beginning of the flowering process and greater efficiency and reduced costs could 
perhaps be achieved by apportioning more of the light energy to crops at this growth 
stage. It was also shown that by using lamps to provide irradiance levels of 75 W m" 2 , 
year-round crops of cannabis could be grown in a totally enclosed environment with no 
natural lighting. These showed no significant difference in yield compared to those 
grown in the glasshouse under supplementary lighting. 

It was found that the yield of botanical raw material produced per unit area was linearly 
proportional to the average irradiance level in that growing environment. Raising the 
irradiance level also caused the plant to divert a higher proportion of energy to the 
biosynthesis of cannabinoids. The amount of cannabinoid produced was therefore 
strongly related to the consumption of electrical lighting energy. In a drive to produce 
flowering cannabis crops with a uniform secondary metabolite content throughout the 
year, irradiance levels were kept as uniform as practicable throughout the twelve hour 
day. In mid-summer, this could mean that supplementary lighting would be used to 
achieve target irradiance levels during dull periods of the day, but on clear days 
glasshouse roof shades would be closed to reduce the ingress of free sunlight around 
noon. This was not necessarily the most energy efficient policy. It is possible that the 
allocation of carbohydrate to primary or secondary metabolite biosynthesis is less 
dependent on the irradiance level (i.e the light energy per unit area per unit time) and 
more closely related to the total quantity of light energy falling on the crop during any 
one twelve hour day - or possible an even longer period. As observed in the study here 
with variegated cannabis, cannabinoid biosynthesis continues in tissue that is not 



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photosynthesising. Indeed Crombie (1977) reported that cannabinoid biosynthesis 
continues during the night, the carbohydrate used therefore having been formed many 
hours earlier. Had uniformity of crop been less important, the glasshouse 
supplementary lighting system could have been switched on continuously on summer 
days and this would have boosted yields of raw material and cannabinoids even 
further, but possibly altered the secondary metabolite profile. The most likely changes 
within the secondary metabolite profile would have been alterations to the ratio of 
monoterpenes and sesquiterpenes, as a consequence of a predicted altering in the 
ratio of foliar and floral material. 

Field trials over several seasons showed that the Sativex-dependent CBD crop could 
also be propagated outdoors in the UK. The costs were greatly reduced but a range of 
problems were encountered, of which fungal infection with Botrytis cinerea caused the 
greatest crop losses. Initially low levels of infection would frequently ruin crops within 
the first 48 hours of the drying process, if plants were dried at 30°C or below. 
Increasing crop drying temperatures up to 50°C considerably reduced crop drying 
times and offered some reduction in the level of fungal spoilage. Over a five year 
period, the crop regularly commenced floral growth in the last ten days of August and it 
was judged ready for harvest in the second week of October, when environmental 
conditions favoured fungal attack. Future outdoor growth appears unlikely without the 
use of fungicides to control disease. Sativex-dependant THC varieties commenced 
flowering too late in September to produce a satisfactory crop, but it was shown that 
other high yielding THC genotypes could produce healthy high-yielding crops outdoors 
in Southern England. 

A major first step in the optimisation of cannabis as a phytopharmaceutical was to 
check that botanical raw material produced from clones was more uniform than that in 
plants grown directly from seeds, even in a highly inbred variety exhibiting pronounced 
phenotypic uniformity. It is common practise amongst licensed and illicit growers to 
grow plants from seed and to make clones from the highest yielding or most potent 
progeny. The plants grown to maturity from these clones typically maintain the 
desirable trait. Research here showed that cloned plants exhibited a significantly more 
uniform cannabinoid profile as well as a higher cannabinoid yield. 

Grown indoors or out, the cannabinoid and terpene profiles were shown to change 
throughout the female floral development stages, with both tending to stabilise once the 
formation of new florets within the inflorescence ceased. The near or complete 
absence of newly formed stigmas was a visible conformation of the readiness of plants 
for harvest. Grown in tightly controlled indoor conditions, this growth stage could be 



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Chapter 7 General Discussion 



routinely achieved after a fixed time and harvest times could be planned in advance. In 
outdoor growing conditions plant development was under strict photoperiodic control. 
Over five consecutive seasons crops of the CBD chemovar G5 were seen to 
commence flowering within a nine day period between the 22 nd August and 1 st 
September, when daylength was approximately 14 hours. In all trials the crop finished 
flowering in the second week of October, just two weeks after the autumnal equinox, 
and this date could be predicted as optimum for future harvests of this variety at this 
latitude. Studies of the cannabinoid profile and terpene profile in both environments 
showed that by the end of the flowering period the cannabinoid and terpene profiles 
were most stable and the proportion of CBG within the cannabinoid profile was at a 
minimum. This was the ideal time to harvest the plant for economic and quality 
reasons. The proportion of propyl (THCV, CBDV etc.) and pentyl side-chained 
cannabinoids (THC, CBD etc.) were seen to change through the flowering period and 
hence the ratio of these would be affected by growth stage at harvest. 

As recommended in many growing guides, illicit cannabis is typically induced to flower 
by exposing it to a steady twelve hour daylength. In an outdoor setting this is the 
daylength naturally occurring at the autumn equinox when outdoor grown crops were 
seen to finish flowering. The CBD chemovar, grown outdoors, was seen to commence 
flowering when daylength fell to approximately fourteen hours per day, and this would 
be defined as its 'critical daylength'. At this point a phytochrome-controlled hormone 
release mechanism induces flowering. The use of a twelve hour day in the glasshouse 
is thus shorter than that required to induce flowering in this chemovar and denies a 
flowering crop access to an additional two hours light exposure and associated 
photosynthetic activity. This in turn might reasonably be expected to affect raw material 
and cannabinoid yields. However, glasshouse studies showed this prediction to be 
incorrect. The studies in question compared the development of plants grown in 
eleven, twelve or thirteen hour daylengths. In all regimes the plants were observed to 
commence flowering equally quickly. However, those maintained in thirteen hour days 
were slower to cease vegetative growth. This resulted in significant changes in the 
cannabinoid profile, a significant increase in height and no increase in the feedstock 
yield, despite a proportional increase in electrical lighting energy costs. This finding 
suggests that there is more than one critical daylength in cannabis, one of which 
induces flowering and a shorter daylength at which vegetative growth is hormonally 
inhibited. Further reducing the daylength below twelve hours resulted in significant 
decreases in raw material and cannabinoid yield. Consequently, it is recommended 



181 



Chapter 7 General Discussion 



that a twelve hour daylength should continue to be used as the standard daylength in 
which to maintain the cannabis crops through the flowering stage. 

Looking ahead, the survey of the cannabinoid content of illicit cannabis in the UK in 
2005 was clearly 'a snapshot in time'. The results showed that the potency and market 
share of cannabis resin, herbal cannabis and sinsemilla were changing and this 
warranted a future study to monitor these changes. Indeed, such a survey was 
performed by the Home Office Scientific and Development Branch in 2008, and 
acknowledged assistance was given (Hardwick and King 2008). A further Home Office 
study is planned to monitor on-going changes in the potency of street cannabis in late 
2009. 

Within the GW Pharmaceuticals glasshouse a number of additional studies are 
foreseen. More research is recommended to investigate ways of further improving the 
winter and summer yield of glasshouse grown crops by alterations to light energy 
levels. In addition to placing greater emphasis on total the light energy received by the 
plant, rather than the irradiance conditions prevailing, future studies should be 
performed to research the correlation between the total quantity of light energy 
received and the relative allocation of carbohydrate to primary or secondary metabolite 
biosynthesis. The observation that high irradiance levels appear to have greatest 
impact on cannabinoid yields when applied at the beginning of flowering requires 
further investigation. If confirmed, supplementary lighting may be selectively 
concentrated on plants at this growth stage. The analytical studies, in which terpene 
profiles were monitored during plant development, should be repeated in view of the 
small number of crops assessed to date. 

To enable CBD chemovar crops to be cultivated outdoors, the application of fungicides 
seems increasingly likely. A range of fungicides should be tested and residues within 
the crop assessed. Alternatively, a more southerly growing location should be 
evaluated. Plant breeding of earlier flowering varieties may overcome disease 
problems, but it may be impossible to breed such varieties with sufficiently similar 
cannabinoid and terpene profiles to be regarded as being of the same chemotype. 
Rather than relying on a mixed feedstock of dried foliar and floral material, the 
collection and storage of enriched trichome preparations offers distinct advantages. 
Further evaluation of trichome collection methods is highly recommended. The 
technique is already performed on an industrial scale with the closest relative genus - 
Humulus (the hop) and further work is recommended to see if the available machinery 
could be adapted for use on cannabis. 



182 



Chapter 7 General Discussion 



As the site of the biosynthesis of cannabinoids, monoterpenes and sesquiterpenes, the 
glandular trichomes are arguably the most important part of the plant. A greater 
understanding of the activities within these trichomes would possibly enable the grower 
to identify when these structures were operating at their optimum. The grower would 
then be able to assess how difference in growing conditions altered the level of 
activities within the trichome. As part of a general study of these structures, the 
capitate stalked form was seen to be the most important type, producing the bulk of the 
cannabinoids on female floral material. Using relatively simple light microscopes, the 
study showed that the terpenoids are sequestered in a resin head. Within a mature 
resin head a disk of secretory cells is visible, occupying about 20% of the volume. Vital 
stains were perceived as a tool to locate and highlight those tissues where respiratory 
activity was taking place within the trichome, and to indicate when this was most active. 
One such stain 'tetrazolium red' suggested that almost all the metabolic activity took 
place within the secretory cells, even though the site of cannabinoid biosynthesis is 
purported to be within the storage space above the cells (Sirikantaramas et ai, 2005, 
Taura et ai, 2007). The secretory cells would indeed consume much energy in the 
biosynthesis of cannabinoid precursors and cannabinoid synthase enzyme. However, 
the lack of apparent activity within the storage space may be purely an anomaly, due to 
the inability of these water based stains to penetrate this area. The use of lypophylic 
alternatives may improve the assessment of metabolic activity within the secretory 
head. 

Enriched trichome preparations, almost entirely containing capitate stalked trichome 
resin heads, were found to have a wide range of cannabinoid contents from 
approximately 30% up to more than 67% % w/w (dry). This may be due to varying 
quantities of membrane tissue, enzymes and terpenes. If enriched trichome 
preparations were to be used as phytopharmaceutical feedstock, rather than floral and 
foliar material, it would be important to gain further understanding of the cause of this 
variability. 

In September 2008, GW Pharmaceuticals pic reported positive results from a placebo- 
controlled randomized withdrawal study of Sativex in patients with neuropathic pain 
due to MS. In February 2009 the company reported positive results in a similar trial with 
patients experiencing spasticity due to MS. These studies showed that the efficacy of 
Sativex in the treatment of both neuropathic pain and spasticity due to MS is 
maintained in long-term use. A few days before completion of thesis GW 
Pharmaceuticals pic announced positive results from a pivotal Phase III double-blind 
randomised placebo-controlled study of Sativex® in patients with spasticity due to MS, 



183 



Chapter 7 General Discussion 



who have achieved inadequate spasticity relief with existing therapies. This Phase III 
study used an enriched design whereby 573 patients initially received Sativex for four 
weeks in a single blind manner (Phase A), following which Sativex responders (n = 
241) were randomized to continue on Sativex or switch to placebo for a further twelve 
weeks in a double-blinded manner (Phase B). The prospectively defined primary 
efficacy endpoint of the study - the difference between the mean change in spasticity 
severity of Sativex vs Placebo in Phase B - was highly statistically significantly in favour 
of Sativex (p = 0.0002). The difference between Sativex and placebo was also 
significant for a number of secondary endpoints. 74% of Sativex patients achieved an 
improvement of greater than 30% in their spasticity score over the entire study versus 
51% on placebo (p = 0.0003). These three studies was performed following regulatory 
guidance from the UK regulatory authority (MHRA) and provided evidence of long term 
efficacy to be included as part of a forthcoming European regulatory submission 
planned for mid 2009. If successful this would require a large increase in the quantity 
of crop grown. The research performed for this thesis aids the reliable propagation of 
that crop. In the UK alone, MS is the most common disabling neurological disease of 
young adults, affecting approximately 85000 people. Approximately 2% of the entire 
UK population experiences neuropathic pain. Regulatory approval of Sativex® would 
lead to the medicine being widely available on prescription. For many of these 
patients, illicit cannabis would have been the only form of medicinal cannabis available 
hitherto. 

In early 2009, the company was performing Phase I trials to evaluate the cannabinoid 
THCV for appetite control in treating obesity. Other cannabinoids were also undergoing 
a range of in-vitro and in-vivo studies. A range of different Cannabis chemovars had 
been specifically bred to be dominant in cannabinoids other than THC and CBD. The 
parent plants used to breed these chemovars differed in provenance, and the optimum 
growing conditions for these had not been determined as fully as those used to 
produce THC and CBD. More horticultural research would be required to optimise their 
propagation. 

This thesis has emphasised many times the fundamental importance of the glandular 
trichome on Cannabis sativa L. It is here, within the vesicles of the glandular trichome 
that the enzymes and precursors of cannabinoid biosynthesis are secreted and the 
phytocannabinoid story starts. Further research will aim to improve the understanding 
of the biosynthetic activities within these trichomes and take this story forward. 



184 



References 



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214 



Appendix 8.1 Analysis of cannabinoid content using HPLC and GC 



Appendix 8.1 Analysis of cannabinoid content using HPLC 

and GC 



8.1.1 HPLC Analysis 




8.1.1.1 HPLC and GC Equipment 




Apparatus 


Source 


0/M-iinn4rtr IV /-I I I ID OO^T 

oonicaior iviouei uk-oz4 i 


Gemini B.V., Elsweg 57, 

7311 GV, Apeldoorn, Netherlands 


i_i*-ii.fif-i44 D^#-jx^»-*-i /a *^;iyn-.+ a <inn udi 

Hewlett Packard/Agilent 1 iuu HrLO 
incorporating: - 

Autosampler DE594901 1 33 


Agilent Technologies UK Ltd. 
South Queensferry, 
West Lothian. 


Diode Ray Detector DE91 605830 




Column Oven DE91609412 




Mobile Phase Degaser Unit DE9605830 




Discovery C8 150 x 4.6 mm column and 
30 x 4.6 mm precolumn packed with 5pm 
Kingsorb ODS packing material. 




Personal computer with HP Chemstation 
software. 




Snniratnr Mnrlpl UR-??4T 


Gpmini R V Fkwpn *S7 7?11 

uci i iii ii lj.v.j i low cy \J 1 j 1 \J I 1 

GV, Apeldoorn, Netherlands 


Hewlett Packard 6890 GC with FID incorporating 
an HP-5 320 pm x 30 m column with a 0.25 pm 
film and an autoanalyser 


Pre- owned item 


Table 8.1.1 Analytical Equipment 




215 





Appendix 8.1 Analysis of cannabinoid content using HPLC and GC 



8.1.1.2 Analytical Materials 


Materials 


Source 


1 1 -Nor-A 9 -tetrahydrocannabinol-9- 
carboxylic acid 50 |jg/ml_ in methanol. 


Sigma Aldrich Ltd., Fancy Road, Poole, 
Dorset 


CBGA 

ObUA 

CBCA 


Applied Analysis Ltd. Rowley House, 
Tokenspire Business Park, 
Beverley, Yorkshire. 


Chloroform HPLC Grade 


Ultrafine Ltd. Marlborough House, 298 
Regents Park Road, London 


Methanol HPLC Grade 


Fisher Scientific UK Ltd. Bishops 
Meadow Lane, Loughborough, Leics. 


Table 8.1.2 Analytical Materials 



8.1.2 HPLC determination of cannabinoid content 



Previous forensic analysts have reported that investigations into cannabis potency are 
made difficult by the inhomogeneous nature of herbal cannabis. Within a well-mixed 
single large batch of crude material, and following removal of unwanted matter, 
different aliquots could lead to quite different analytical results (King 2004). Batches of 
the 'naturally inhomogeneous material' were well mixed before sampling. A minimum of 
three subsamples was analysed if sufficient material was available. 

The botanical raw material is thoroughly mixed. Five small samples of approximately 1 
g are taken at random from the mixture and blended. From this, a single sub-sample of 
100 mg is extracted with 1.0 ml methanol-chloroform (9:1 v/v) by sonication for fifteen 
minutes. 100 pi of the filtered extract is diluted with 300 pi of methanol and aliquots of 
1 pi used for HPLC. 

Using a Discovery C8 150 x 4.6 mm column and a 30 x 4.6 mm precolumn containing 
5pm Kingsorb ODS packing material, an operating temperature of 25°C and a UV 
wavelength of 220 nm were adopted. The run time was 23 minutes and THC the 
internal standard. 

Apart from THC, which was purchased from Sigma-Aldrich, most cannabinoid 
analytical standards were not available at the commencement of this study. 
Cannabinoids such as CBGA were identified and quantified in the HPLC trace by 



216 



Appendix 8.1 Analysis of cannabinoid content using HPLC and GC 



comparing chromatograms with those produced by Lehmann and Brenneisen (1995). 
CBGA, CBDA and CBCA were later purified and identified at Applied Analysis Ltd 
(Flockhart pers comm.) and the original identification of their HPLC peaks confirmed. 

A chromatogram produced when analysing an extract of Clone Line G1 M1 is shown in 
Figure 8.1.1. The CBGA and THCA peaks are observed at 6.56 and 15.45 minutes 
respectively. Following the later acquisition of a CBGA standard it was ascertained 
that when equimolar preparations of THCA and CBGA are evaluated by HPLC using 
this method, CBGA produces a peak area greater than THCA by a factor of 1.2. 
Concentrations of the two in this thesis have been calculated accordingly. Corrections 
for the peak characteristics of other cannabinoids have been similarly implemented. 



217 



Appendix 8.1 Analysis of cannabinoid content using HPLC and GC 




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Figure 8.3.1 Chromatogram of a preparation from Clone G1 M1. 



218 



Appendix 8.1 Analysis of cannabinoid content using HPLC and GC 



8.1.3 GC determination of cannabinoid content 

The botanical raw material was thoroughly mixed. Five small samples are normally 
taken at random from the mixture and blended. From this, a single sample 50 mg was 
taken and prepared for analysis. 

The samples were then prepared and analysed as developed by de Meijer et al 2003. I 
ml of ethanol (>99.7%) was added to the filtration tube and the sample sonicated for 15 
minutes and the extract then centrifuged at 4000 rpm for 10 minutes. This procedure 
was then repeated a further three times and the resultant 4 ml of ethanol containing the 
cannabinoid extracts then transferred to a five ml volumetric flask. 0.25 ml of a 
phenanthrene stock solution (10mg/ml) in ethanol was added as an internal standard 
and adjusted to 5 ml with ethanol. Extracts were homogenised and transferred to GC 
vials. 

Gas-chromatographic analyses were performed on a Hewlett Packard 6890 GC 
equipped with an autoanalyser, a flame ionization detector and an HP-5 320 urn x 30 m 
column with a 0.25 urn film. 



219 



Appendix 8.2 Analysis of terpene content using GC 



Appendix 8.2 Analysis of terpene content using GC 



8.2. 1 Analysis Equipment 



Apparatus 


Source 


Gas Chromatograph with split/splitless capillary 
injector (Agilent Technologies 5890 or 6890) 

Flame ionisation detector (FID) and/or Electron 
lonisation Mass Spectrometer (EI-MS) 
Autosampler DE594901 1 33 

Diode Ray Detector DE91 605830 

Column Oven DE91609412 

Mobile Phase Degaser Unit DE9605830 

Discovery C8 150 x 4.6 mm column and 

30 x 4.6 mm precolumn packed with 5pm Kingsorb 

ODS packing material. 

Personal computer with HP Chemstation software. 


Agilent Technologies UK Ltd. 
South Queensferry, 
West Lothian. 



Table 8.2.1 Analytical equipment 



8.2.2 GC Analysis Method 

The botanical raw material was thoroughly mixed. 1 g samples are taken at random 
and extracted with approximately 40 ml of methanokchloroform (9:1 v/v) in a 50 ml 
volumetric flask by sonication for thirty minutes at 25°C. The solution is allowed to cool 
and the volume made up to 50ml with methanokchloroform (9:1 v/v). An aliquot of each 
extract is centrifuged at 3000 rpm for 2 minutes. 1 .5ml of the supernatant is added to a 
2ml auto-sampler vial, capped and securely crimped prior to analysis. Injector and 
Detector temperatures are 250°C and 325°C respectively. The injection volume is 1 pi 
with a split ration of 5:1. The FID fuel gases were Hydrogen 40 ml min-1, Air 50ml 
min" 1 , Helium 45 ml min" 1 . 



220 



Appendix 8.2 Analysis of terpene content using GC 



Groups of terpenes may be identified at retention times of approximately 4-5 minutes 
for monoterpenes and 14-20 minutes for sesquiterpenes. Peak identifications may be 
made by retention time comparisons with certified standards and by using gathered 
mass spectra. For generation of accurate quantitative data the losses and uncertainties 
of the injection and volatilisation process can be reduced by using an internal standard 
- Phenanthrene at 1 mg ml" 1 

The assay methods described in the above two appendices have been validated in 
accordance with ICH Guidelines and have subsequently been included in product 
license applications. The exact details of this validation remain the intellectual property 
of GW Pharmaceuticals Ltd. 



221 



Appendix 8.3 A Comparison of the Structure of CB[ and CB 2 Receptors 




a) 




b) 

The structure of CB1 (a) and CB2 receptors (b) in human tissue. Above the cell 
membrane is the extra-cellular N-terminal and below the intracellular C-terminal. 



222 



Appendix 8.4 Meteorological Data pertaining to UK Field Trials 



Appendix 8.4 Meteorological Data pertaining to UK Field 

Trials 





2000 


2003 


2004 


2005 


2006 


Mean 


May 


12.7 


12.2 


12.5 


11.8 


8.4 


11.5 


Jun 


15.3 


16.3 


15.9 


16.0 


16.2 


15.9 


Jul 


15.7 


17.9 


16.5 


17.3 


20.3 


17.5 


Aug 


17.2 


19.2 


18.0 


16.7 


16.9 


17.6 


Sep 


15.3 


14.7 


15.4 


16.0 


17.3 


15.7 



8.4.1 Mean daily temperatures in the field trial region as recorded by the Meteorological 
Service. 





2000 


2003 


2004 


2005 


2006 


Mean 


May 


196 


202 


206 


224 


168 


192 


Jun 


174 


224 


232 


215 


274 


224 


Jul 


174 


206 


189 


202 


311 


216 


Aug 


215 


240 


202 


242 


181 


216 


Sep 


128 


194 


173 


158 


164 


163 



8.4.2 Total number of sunshine hours each month in the field trial region as reported by the 
Meteorological Service. 



The above data was downloaded from the Meteorological Office web page: - 
http://www.metoffice.gov.uk/climate/uk [accessed on 6th February 2009]. 



223 



Appendix 8.5 Definitions of Social Grade as defined by National Readership Survey 



Appendix 8.5 Definitions of Social Grade as defined by 
National Readership Survey 

The Social Grade classification used by NRS was developed for the Survey over 50 
years ago. It remains a highly effective way of classifying readers of different 
publications, and is widely used in planning advertising, not just in newspapers and 
magazines, but in other media too. The grades were also used by the Government 
when compiling the UK Population Census. 

Social Grade is determined by the occupation of the Chief Income Earner (CIE) in each 
household. Additional criteria such the size of the organisation, and the number of 
people for which the CIE is responsible, are used to refine the process. 

A brief description of the six grades is as follows: 



Social Status 

A Upper Middle Class 
B Middle Class 

C1 Lower Middle Class 

C2 Skilled Working Class 
D Working Class 



Occupation 

Higher managerial, administrative or professional 

Intermediate managerial, administrative or professional 

Supervisory or clerical and junior managerial, 
administrative or professional 

Skilled manual workers 

Semi and unskilled manual workers 



Those at the lowest levels Casual or lowest grade workers, pensioners and others 



of subsistence 



who depend on the state for their income 



224